WO2023210661A1 - COMPOSITION POUR RÉGULER L'ACTIVATION DES CELLULES CAR-T, LA PROLIFÉRATION DES CELLULES SOUCHES HÉMATOPOÏÉTIQUES ET LA DIFFÉRENCIATION DES CELLULES iPS, ET SON UTILISATION - Google Patents

COMPOSITION POUR RÉGULER L'ACTIVATION DES CELLULES CAR-T, LA PROLIFÉRATION DES CELLULES SOUCHES HÉMATOPOÏÉTIQUES ET LA DIFFÉRENCIATION DES CELLULES iPS, ET SON UTILISATION Download PDF

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WO2023210661A1
WO2023210661A1 PCT/JP2023/016351 JP2023016351W WO2023210661A1 WO 2023210661 A1 WO2023210661 A1 WO 2023210661A1 JP 2023016351 W JP2023016351 W JP 2023016351W WO 2023210661 A1 WO2023210661 A1 WO 2023210661A1
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membrane
protein
extracellular vesicle
extracellular
cell
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PCT/JP2023/016351
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Japanese (ja)
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力成 華山
友義 山野
一隆 的場
克彦 木田
志保 阿武
泰斗 西野
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国立大学法人金沢大学
日産化学株式会社
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A61P7/06—Antianaemics
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
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    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
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    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
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    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material

Definitions

  • the present invention relates to a composition that controls CAR-T cell activation, hematopoietic stem cell proliferation, and iPS cell differentiation, and uses thereof.
  • Antigen-specific T cells e.g., cytotoxic T cells, helper T cells, etc.
  • Antigen-specific T cells play a central role in immune responses such as the elimination of cancer cells by living organisms and the regulation of responses to self-antigens, allergic substances, etc. It is known to have a similar function.
  • Antigen-specific T cells use their T cell receptors to recognize binding complexes between MHC molecules on the cell surface of antigen-presenting cells such as dendritic cells and macrophages and antigens derived from cancer, allergic substances, etc. activation, proliferation, differentiation, etc.
  • Activated antigen-specific T cells specifically damage antigen-presenting cancer cells, etc., and regulate responses to self-antigens, allergic substances, etc. Therefore, activation, proliferation, differentiation, etc. of antigen-specific T cells are considered to be particularly important in immune reactions.
  • Patent Document 1 discloses that nanoparticles containing MHC molecules and T cell costimulatory molecules on their surface proliferate antigen-specific T cells. Furthermore, Non-Patent Document 1 discloses that exosomes in which IL-12 is expressed on the membrane via PTGFRN proliferate model antigen-specific CD8-positive T cells.
  • the present inventors have also disclosed a method for activating various T cells using exosomes containing antigen-presenting MHC molecules and T cell costimulatory molecules on their surfaces (Patent Documents 2 and 3).
  • the purpose of the present invention is to provide a novel cell activation method, a composition for cell activation, and uses thereof, which activate specific cells (i.e., proliferate and/or differentiate) more efficiently than co-administering cytokine molecules. .
  • iPS cells iPS cells
  • iPS cells can be activated (i.e., proliferated and/or differentiated).
  • the invention includes: [0] Extracellular vesicles that extracellularly present at least one cytokine. [1] Extracellular vesicles that present at least one target factor extracellularly. [2] Extracellular vesicles that extracellularly present at least one target factor and at least one cytokine.
  • [1A] The extracellular vesicle according to [2], wherein the target factor is an antigen and the cytokine is a T cell-stimulating cytokine.
  • [2A] The extracellular vesicle according to [2], wherein the target factor is an antigen and a T cell costimulatory molecule, and the cytokine is a T cell stimulating cytokine.
  • Possible proteins or (2) (C) an extracellular vesicle containing the antigen and a protein capable of extramembranely presenting the antigen and the T cell-stimulating cytokine, including the T cell-stimulating cytokine or a subunit thereof.
  • [5A] The extracellular vesicle according to [2A], the membrane of which has the following: (1) (B) A protein containing the antigen and capable of presenting the antigen outside the membrane; (A) a protein capable of extramembranely presenting the cell-stimulating cytokine, including the T-cell-stimulating cytokine or a subunit thereof; and (D) a protein capable of presenting the T-cell costimulatory molecule, including the T-cell costimulatory molecule.
  • Protein (B) is antigen and Any one of [3A] to [5A], which is a fusion protein with a membrane protein capable of localizing in the membrane of an extracellular vesicle or a protein capable of binding to the membrane of an extracellular vesicle. Extracellular vesicles as described in.
  • Protein (A) is the T cell-stimulating cytokine or a subunit thereof; The extracellular according to [4A] or [5A], which is a fusion protein with a membrane protein capable of localizing in the membrane of an extracellular vesicle or a protein capable of binding to the membrane of an extracellular vesicle. vesicle.
  • Protein (C) is the antigen; the T cell-stimulating cytokine or a subunit thereof;
  • vesicle. [9A]
  • the membrane protein capable of localizing in the membrane of an extracellular vesicle or the protein capable of binding to the membrane of an extracellular vesicle comprises a tetraspanin or its transmembrane domain or MFG-E8 or its membrane binding domain.
  • Protein (C) is From the N-terminal side, (C-1) antigen peptide, (C-2) a spacer sequence that may be present; and (C-3) a fusion comprising a tetraspanin or its transmembrane domain or MFG-E8 or its transmembrane domain and the T cell-stimulating cytokine or its subunit.
  • the extracellular vesicle according to [4A] or [5A] which comprises an amino acid sequence encoding a peptide in this order.
  • Protein (C) is From the N-terminal side, (C-1) A fusion peptide comprising a tetraspanin or its transmembrane domain or MFG-E8 or its transmembrane domain and the T cell-stimulating cytokine or its subunit (C-2) a spacer sequence that may be present, and (C-3) antigen peptide,
  • C-1 A fusion peptide comprising a tetraspanin or its transmembrane domain or MFG-E8 or its transmembrane domain and the T cell-stimulating cytokine or its subunit
  • C-3 antigen peptide The extracellular vesicle according to [4A] or [5A], which contains the amino acid sequence encoded in this order.
  • [12A] The fusion peptide, from the N-terminal side, (1) A partial sequence of a tetraspanin comprising transmembrane domain 1, small extracellular loop, transmembrane domain 2, small intracellular loop and transmembrane domain 3, (2) a spacer sequence that may be present; (3) the T cell-stimulating cytokine or a subunit thereof; (4) an optional spacer sequence, and (5) an amino acid sequence encoding a partial sequence of tetraspanin containing transmembrane domain 4 in this order, the extracellular small according to [10A] or [11A].
  • Cell A partial sequence of a tetraspanin comprising transmembrane domain 1, small extracellular loop, transmembrane domain 2, small intracellular loop and transmembrane domain 3, (2) a spacer sequence that may be present; (3) the T cell-stimulating cytokine or a subunit thereof; (4) an optional spacer sequence, and (5) an amino acid sequence encoding a partial
  • [13A] The fusion peptide, from the N-terminal side, (1) the T cell-stimulating cytokine or a subunit thereof; (2) a spacer sequence that may be present, and (3) MFG-E8
  • the fusion protein defined in (D) above comprises a T cell costimulatory molecule; [ 5A].
  • [15A] The fusion protein defined in (D) above comprises a T cell costimulatory molecule;
  • the extracellular vesicle according to [5A] comprising a tetraspanin or its transmembrane domain, or MFG-E8 or its membrane binding domain.
  • [16A] From the N-terminal side of the fusion protein defined in (D) above, (D-1) Amino acid sequence of T cell costimulatory molecule, (D-2) The extracellular vesicle according to [5A], comprising an optional spacer sequence, and (D-3) an amino acid sequence comprising the amino acid sequence of a tetraspanin or a transmembrane domain thereof in this order. [17A] The extracellular vesicle according to any one of [0A] to [16A], wherein the extracellular vesicle is an exosome.
  • [1AA] (a) A polynucleotide encoding the protein (A) defined in [4A]; (b) a polynucleotide encoding the protein (B) defined by [3A] or [4A]; (c) a polynucleotide encoding the protein (C) defined in [4A]; (d) A polynucleotide encoding the protein (D) defined in [5A]; or (e) A polynucleotide encoding the protein (E) defined in [5A]. [2AA] A vector comprising the polynucleotide described in [1AA].
  • [3AA] (a) a polynucleotide encoding a protein (A) as defined in [4A]; and (b) a polynucleotide encoding a protein (B) as defined in [3A] or [4A].
  • [4AA] (c) A cell transformed with a vector containing a polynucleotide encoding protein (C) defined in [5A].
  • [5AA] (d) The cell according to [3AA] or [4AA], further comprising a polynucleotide encoding the protein (D) defined in [5A].
  • [6AA] (e) A cell transformed with a vector containing a polynucleotide encoding the protein (E) defined in [5A].
  • [7AA] A culture supernatant obtained by culturing the cells according to any one of [3AA] to [6AA].
  • [8AA] Extracellular vesicles contained in the culture supernatant according to [7AA].
  • [9AA] A method for producing the extracellular vesicle according to [1A], comprising: 1) culturing the cells according to [3AA] or [4AA], 2) collecting the culture supernatant after culturing; and 3) optionally purifying extracellular vesicles from the collected culture supernatant.
  • [10AA] A method for producing the extracellular vesicle according to [2A], comprising: 1) culturing the cells according to [5AA] or [6AA], 2) collecting the culture supernatant after culturing; and 3) optionally purifying extracellular vesicles from the collected culture supernatant.
  • [1AB] A pharmaceutical composition comprising the extracellular vesicle according to any one of [0A] to [3A].
  • [2AB] A pharmaceutical composition for proliferating chimeric antigen receptor gene-transferred T cells (CAR-T cells) specific for the antigen in vivo or in vitro, the composition according to any one of [0A] to [3A].
  • a pharmaceutical composition comprising an extracellular vesicle as described.
  • [3AB] A pharmaceutical composition for treating cancer containing cancer cells expressing the antigen, comprising the extracellular vesicle according to any one of [0A] to [3A], A pharmaceutical composition that is administered to a patient who has received chimeric antigen receptor gene-transferred T cells (CAR-T cells).
  • [4AB] The pharmaceutical composition according to [2AB] or [3AB], wherein the antigen is Her2 protein or a fragment thereof, or CD19 protein or a fragment thereof.
  • [1AC] A method for activating and/or proliferating CAR-T cells in a subject to which the antigen-specific chimeric antigen receptor gene-transferred T cells (CAR-T cells) have been administered, the method comprising: ] to [3A], comprising administering the extracellular vesicle according to any one of [3A],
  • the chimeric antigen receptor of the CAR-T cell reacts with the antigen presented on the extracellular membrane of the extracellular vesicle, and preferably, the T cell-stimulating cytokine receptor on the CAR-T cell and the extracellular vesicle react with each other.
  • the T cell-stimulating cytokines presented on the extracellular membrane of the vesicle react, and preferably, CD28, CD134, etc. present on the membrane of the CAR-T cell and the T cells presented on the extracellular membrane of the extracellular vesicle react with each other.
  • the chimeric antigen receptor of the CAR-T cell reacts with the antigen presented on the extracellular membrane of the extracellular vesicle, and preferably, the T cell-stimulating cytokine receptor on the CAR-T cell and the extracellular vesicle react with each other.
  • the T cell-stimulating cytokines presented on the extracellular membrane of the vesicle react, and preferably, CD28, CD134, etc. present on the membrane of the CAR-T cell and the T cells presented on the extracellular membrane of the extracellular vesicle react with each other.
  • CAR-T cells Due to the interaction of the stimulating molecules, CAR-T cells are activated and/or proliferated within the subject, and the activated and/or proliferated CAR-T cells attack the cancer cells, resulting in cancer cells.
  • a method for treating cancer by suppressing the proliferation of. [3AC] The method according to [1AC] or [2AC], wherein the antigen is Her2 protein or a fragment thereof, or CD19 protein or a fragment thereof.
  • [1AD] In the production of a medicament for activating and/or proliferating CAR-T cells in a subject to whom the antigen-specific chimeric antigen receptor gene-transferred T cells (CAR-T cells) have been administered, [0A] to [0] 3A].
  • [2AD] Use of the extracellular vesicle according to any one of [0A] to [3A] in the manufacture of a medicament for treating cancer containing cancer cells expressing the antigen in a subject, the use of the extracellular vesicle according to any one of [0A] to [3A], Use in which specific chimeric antigen receptor transgenic T cells (CAR-T cells) are administered to the subject.
  • [3AD] The method according to [1AD] or [2AD], wherein the antigen is Her2 protein or a fragment thereof, or CD19 protein or a fragment thereof.
  • TPO thrombopoietin
  • SCF stem cell factor
  • [4B] (B) The extracellular small according to any one of [1B] to [3B], which contains L-selectin and/or CXCL12 and further contains a protein capable of extramembrane display of L-selectin and/or CXCL12.
  • Protein (A)-1 is a fusion protein of TPO and a membrane protein capable of localizing in the membrane of extracellular vesicles or a protein capable of binding to the membrane of extracellular vesicles, [ 2B].
  • Protein (A)-2 is a fusion protein of SCF and a membrane protein capable of localizing in the membrane of extracellular vesicles or a protein capable of binding to the membrane of extracellular vesicles, [ 2B].
  • Protein (A)-3 is The TPO; The SCF; The extracellular vesicle according to [3B], which is a fusion protein with a membrane protein capable of localizing in the membrane of the extracellular vesicle or a protein capable of binding to the membrane of the extracellular vesicle.
  • Protein (B) is The L-selectin or CXCL12; The extracellular vesicle according to [4B], which is a fusion protein with a membrane protein capable of localizing in the membrane of an extracellular vesicle or a protein capable of binding to the membrane of an extracellular vesicle.
  • the membrane protein capable of localizing in the membrane of an extracellular vesicle or the protein capable of binding to the membrane of an extracellular vesicle comprises a tetraspanin or its transmembrane domain or MFG-E8 or its membrane binding domain.
  • [11B] (a)-1 A polynucleotide encoding protein (A)-1 defined in [2B]; (a)-2 A polynucleotide encoding protein (A)-2 defined in [2B]; (a)-3 A polynucleotide encoding protein (A)-3 defined in [3B]; or (b) A polynucleotide encoding protein (B) defined in [4B].
  • [12B] A vector comprising the polynucleotide described in [11B].
  • [13B] (a)-1 A polynucleotide encoding protein (A)-1 defined in [2B]; and/or (a)-2 A polynucleotide encoding protein (A)-2 defined in [2B] Cells transformed with a single vector or a combination of two or more vectors, including: [14B] (a)-3 A cell transformed with a vector containing a polynucleotide encoding protein (A)-3 defined in [3B]. [15B] (d) The cell according to [13B] or [14B], further comprising a polynucleotide encoding the protein (B) defined in [4B].
  • [16B] A culture supernatant obtained by culturing the cells according to any one of [13B] to [15B].
  • [17B] Extracellular vesicles contained in the culture supernatant according to [16B].
  • [18B] A method for producing the extracellular vesicle according to [1B], comprising: 1) culturing the cell according to any one of [13B] to [15B], 2) collecting culture supernatant after culturing; and 3) optionally purifying extracellular vesicles from the collected culture supernatant.
  • [19B] A pharmaceutical composition comprising the extracellular vesicle according to [1B] or the culture supernatant according to [16B].
  • [20B] A pharmaceutical composition for activating and/or proliferating hematopoietic stem cells in vivo or in vitro, comprising the extracellular vesicle according to [1B] or the culture supernatant according to [16B].
  • [21B] A pharmaceutical composition for treating aplastic anemia in a subject, comprising the extracellular vesicles according to [1B] or the culture supernatant according to [16B], and which is administered to a subject who has received hematopoietic stem cells.
  • a pharmaceutical composition A pharmaceutical composition.
  • [22B] A pharmaceutical composition for treating blood cancer or immunodeficiency in a subject, the composition comprising the extracellular vesicle according to [1B] or the culture supernatant according to [16B], wherein the subject receives chemotherapy and A pharmaceutical composition in which hematopoietic stem cells are administered after/or a radiotherapy treatment.
  • [23B] A method for activating and/or proliferating hematopoietic stem cells in a subject who has been administered hematopoietic stem cells, the method comprising administering to the subject the extracellular vesicles described in [1B] or the culture supernatant described in [16B] including doing;
  • the method according to [22B] wherein the subject suffers from aplastic anemia.
  • [24B] A method for treating blood cancer or immunodeficiency in a subject, the method comprising: Administering hematopoietic stem cells to the subject after chemotherapy and/or radiotherapy treatment, and then administering the extracellular vesicles according to [1B] or the culture supernatant according to [16B],
  • the cytokine receptors on the administered hematopoietic stem cells and the cytokines presented outside the membrane of extracellular vesicles react, and the hematopoietic stem cells are activated and/or proliferate within the subject, resulting in hematopoietic function in the subject. How to recover.
  • [25B] Use of the extracellular vesicle according to [1B] or the culture supernatant according to [16B] in the manufacture of a medicament for activating and/or proliferating hematopoietic stem cells in a subject to whom the hematopoietic stem cells have been administered.
  • [26B] The use according to [25B], wherein the subject suffers from aplastic anemia.
  • [27B] Use of the extracellular vesicles according to [1B] or the culture supernatant according to [16B] in the manufacture of a medicament for treating blood cancer or immunodeficiency in a subject,
  • the subject has been administered hematopoietic stem cells after chemotherapy and/or radiotherapy treatment, and cytokine receptors on the administered hematopoietic stem cells and cytokines presented outside the membrane of extracellular vesicles react.
  • [1C] The extracellular vesicle according to [0], wherein the cytokine is ActivinA.
  • [2C] The extracellular vesicle according to [1], wherein the target factor is Bc2Lc and the cytokine is ActivinA.
  • [3C] The extracellular vesicle according to [1C], the membrane of which has the following: (A) An extracellular vesicle containing the Activin A and a protein capable of displaying the Activin A outside the membrane.
  • Protein (B) is a fusion protein of Bc2Lc and a membrane protein capable of localizing in the membrane of extracellular vesicles or a protein capable of binding to the membrane of extracellular vesicles [4C] Extracellular vesicles as described in. [7C]
  • the protein (A) is a fusion protein of Activin A and a membrane protein capable of localizing in the membrane of extracellular vesicles or a protein capable of binding to the membrane of extracellular vesicles [3C] Or the extracellular vesicle according to [4C].
  • Protein (C) is The Bc2Lc and the ActivinA;
  • the extracellular vesicle according to [5C] which is a fusion protein with a membrane protein capable of localizing in the membrane of the extracellular vesicle or a protein capable of binding to the membrane of the extracellular vesicle.
  • the membrane protein capable of localizing in the membrane of an extracellular vesicle or the protein capable of binding to the membrane of an extracellular vesicle comprises a tetraspanin or its transmembrane domain or MFG-E8 or its membrane binding domain.
  • [10C] The extracellular vesicle according to any one of [1C] to [9C], wherein the extracellular vesicle is an exosome.
  • [11C] (a) A polynucleotide encoding a protein (A) defined by [3C] or [4C]; (b) A polynucleotide encoding a protein (B) defined by [4C]; or (c) a polynucleotide encoding a protein (C) defined by [5C].
  • [12C] A vector comprising the polynucleotide described in [11C].
  • [13C] (a) a polynucleotide encoding the protein (A) defined in [3C] or [4C]; and/or (b) a polynucleotide encoding the protein (B) defined in [4C].
  • [14C] (c) A cell transformed with a vector containing a polynucleotide encoding protein (C) defined in [5C].
  • [15C] A culture supernatant obtained by culturing the cells according to any one of [11C] and [12C].
  • [16C] Extracellular vesicles contained in the culture supernatant according to [15C].
  • [17C] A method for producing the extracellular vesicle according to [1C], comprising: 1) culturing the cells according to [11C] or [12C], 2) collecting the culture supernatant after culturing; and 3) optionally purifying extracellular vesicles from the collected culture supernatant.
  • [18C] An agent for inducing differentiation of iPS cells or ES cells, comprising the extracellular vesicle according to [1C] or the culture supernatant according to [13C].
  • MHC Major Histocompatibility Complex
  • Example 1.2.1 Results of flow cytometry analysis of fusion proteins contained in the membrane of extracellular vesicles. Upper row: extracellular vesicles of sample 3; lower row: extracellular vesicles of sample 4.
  • Example 1.3.1 Results of cell proliferation evaluation using human bone marrow CD34-positive progenitor cells.
  • Example 2.2.1 Results of flow cytometry analysis of fusion proteins contained in the membrane of extracellular vesicles.
  • Example 2.3.2. Results of an iPSC differentiation induction experiment using Activin A-expressing extracellular vesicles.
  • Example 3.1 Schematic diagram of each gene structure in . (A) For mouse CAR-T cell experiment.
  • Control exosomes (negative control), hCD19-hCD81, hCD19-hCD81 and hCD80-hCD9, hCD19-hCD81-hIL-2, hCD19-hCD81-hIL-2 and hCD80-hCD9.
  • Extracellular vesicles as used herein are not particularly limited as long as they are vesicles secreted from cells, but examples include Exosomes and Microvesicles (MV). , Apoptotic Bodies, and the like.
  • exosome refers to about 20 to about 500 nm (preferably about 20 to about 200 nm, more preferably about 25 to about 150 nm, even more preferably about 30 to about 100 nm).
  • Components of exosomes include, for example, proteins, nucleic acids (mRNA, miRNA, non-coating RNA), and the like. Exosomes may have the function of controlling intercellular communication. Examples of exosome marker molecules include Alix, Tsg101, tetraspanin, flotillin, and phosphatidylserine.
  • Microvesicles refers to vesicles of about 50 to about 1000 nm that originate from the cytoplasmic membrane.
  • the components of the microvesicle include proteins, nucleic acids (mRNA, miRNA, non-coated RNA, etc.), and the like.
  • Microvesicles can have functions such as controlling intercellular communication.
  • marker molecules for microvesicles include integrins, selectins, CD40, and CD154.
  • apoptotic bodies refers to vesicles of about 500 to about 2000 nm that originate from the cytoplasmic membrane.
  • Components of apoptotic bodies include, for example, fragmented nuclei, organelles, and the like. Apoptotic bodies may have functions such as inducing phagocytosis.
  • marker molecules for apoptotic bodies include Annexin V, phosphatidylserine, and the like.
  • Cytokine refers to a physiologically active protein substance secreted from cells.
  • interferon IFN
  • interleukin IL
  • chemokine CCL, etc.
  • SCF stem cell factor
  • hematopoietic factors colony stimulating factor (CSF), erythropoietin (EPO), thrombopoietin (TPO), etc.
  • TNF tumor necrosis factor
  • growth factors EGF, FGF- ⁇ , etc.
  • activin inhibin, and the like.
  • cytokines may include not only immature (inactive) or mature (active) cytokines, but also partial sequences thereof, subunits of active cytokines.
  • Cytokines may be derived from any animal species. For example, rodents such as mice and rats; lagomorphs such as rabbits; ungulates such as pigs, cows, goats, horses, and sheep; felines such as dogs and cats; humans, monkeys, rhesus monkeys, cynomolgus monkeys, marmosets, Examples include those derived from animals such as mammals such as primates such as orangutans and chimpanzees.
  • the cytokines described herein are preferably of rodent or mammalian origin, more preferably of murine or human origin.
  • the cytokine described herein has an amino acid sequence identity of 80% or more, preferably 90% or more, to its wild type amino acid sequence, as long as it can exert its function. It is preferably 95% or more, more preferably 98% or more, even more preferably 99% or more.
  • the cytokine described herein may have one or more amino acid deletions, insertions, additions, and /or may be substituted.
  • T cell-stimulating cytokine is a cytokine that can stimulate (eg, activate, suppress, etc.) T cells via receptors expressed on the membrane of T cells.
  • T cell stimulating cytokines include, but are not limited to, IL-2, IL-4, IL-6, IL-7, IL-12, IL-15, TGF- ⁇ , IFN- ⁇ , IFN- ⁇ and the like.
  • those that can form multimers of homo or hetero subunits for example, IL-12, TGF- ⁇ , etc.
  • are functional i.e., as long as they can have the desired pharmacological activity).
  • , or may be a continuous amino acid sequence connected via a peptide linker or the like as the case may be. It may be bound or fused to other full-length proteins or partial sequence peptides thereof (eg, the Sushi domain of the IL-15 receptor) as long as the ability to stimulate T cells is maintained.
  • Target factor refers to a molecule that can bind to a molecule present on the surface of a cell that is stimulated by the extracellular vesicle according to the present invention.
  • Molecules present on the surface of cells include, but are not particularly limited to, antibodies, receptors, cell adhesion molecules, sugar chains (and sugar chain proteins), and the like.
  • the molecule present on the cell surface is an antibody, the antigen recognized by the antibody; if the molecule present on the cell surface is a receptor, its ligand (for example, a chemokine such as CXCL12); in the case of a cell adhesion factor, the antigen recognized by the antibody; Molecules that bind to the cell adhesion molecules; if the molecules present on the cell surface are sugar chains (and sugar chain proteins), sugar chain binding proteins that bind to the sugar chains (for example, selectins such as L-selectin and Bc2Lc lectins) etc. correspond to target factors.
  • antigen used herein is not particularly limited as long as it has antigenicity, and includes not only peptidic antigens (i.e., antigenic peptides) but also phospholipids, complex carbohydrates, etc.
  • Non-peptidic antigens such as (eg, bacterial membrane components such as mycolic acid and lipoarabinoannan) are also included.
  • the "antigen peptide” used herein is not particularly limited as long as it is a peptide composed of two or more amino acids (including one composed of more than 50 amino acids) that can serve as an antigen, and is a naturally occurring peptide. It may be derived from natural sources, synthetically derived, or commercially available.
  • the antigenic peptide may include the full-length amino acid sequence of the gene product or a partial amino acid sequence thereof.
  • Antigenic peptides include, but are not limited to, Axl, BAFF-R, B7-H3, BCMA, CAIX, CD19, CD20, CD22, CD38, CD70, CD138, CEA, CLDN6, EpCAM, FAP, Flt3, folate receptor- ⁇ , GD2, Glypican 3, GM-CSF receptor, GRP78, GPC1, HGFR, Integrin ⁇ v ⁇ 6, IL3R, IL13Ra2, TAG72, Mesothelin, MUC1, MUC16, PSCA, PSMA, ROR1, 5T4, WT-1 , ⁇ -fetal protein, MAGE-1, MAGE-3, placental alkaline phosphatase sialyl-Lewis X, CA-125, CA-19, TAG-72, epithelial glycoprotein 2, ⁇ -fetal protein receptor, M2A, tyrosinase, Ras , p53, Her-2/neu, EGF-R, estrogen receptor, progesterone receptor
  • viruses e.g. adenovirus, herpes simplex virus, papillomavirus, respiratory syncytiavirus, poxvirus, HIV, influenza viruses, coronaviruses such as SARS-CoV and SARS-CoV2
  • intracellular parasites e.g. Chlamydiaceae, Mycoplasmatae, Acholeplasmatidae, Rickettsiaceae
  • helminths e.g.
  • Antigenic peptides may include allergens that cause allergic symptoms.
  • allergens include foreign peptides, such as house dust, mites, animals (e.g., companion animals such as cats and dogs), and Examples include peptides derived from pollen (for example, cedar and cypress).
  • a protein (including its full-length sequence and partial sequence) contained in cedar pollen, such as Cryj1 is exemplified.
  • the allergen that causes allergic symptoms may be of food origin.
  • allergens that overcome allergic symptoms to food include peptides derived from chicken eggs, milk, wheat, buckwheat, crab, shrimp, and peanuts (including their full-length sequences and partial sequences).
  • the antigenic peptide may be subjected to any processing or modification (for example, phosphorylation or sugar chain modification).
  • co-stimulatory molecule refers to a secondary signaling molecule that immune cells rely on to activate an immune response upon antigen presentation.
  • the immune cell when the immune cell is a T cell, it refers to a molecule that can contribute to the activation of T cells by interacting with molecules present on the membrane of T cells, such as CD28 and CD134.
  • T cell costimulatory molecules include, but are not limited to, molecules such as CD80 and CD86, or their extracellular domains or functional fragments thereof; anti-CD28 antibodies, anti-CD134 antibodies, etc.
  • antibodies or antigen-binding fragments thereof (e.g., scFv, Fab, nanobody), etc.; fusion proteins (or complexes, aggregates) of these with the transmembrane domain of other proteins, the Fc portion of antibodies, etc. can be mentioned.
  • Extracellular vesicles that display cytokines and/or target factors outside the membrane are those that contain proteins defined in (A) to (E) below in their membranes to display cytokines and/or target factors. may be presented extramembranely. Alternatively, cytokines or target factors may be attached to the membrane surface of the isolated extracellular vesicles afterwards. The attachment method is not particularly limited, but the cytokine or target factor may be attached to the membrane surface by binding a phospholipid to the cytokine or target factor, respectively, and incorporating the new lipid moiety into the membrane of the extracellular vesicle. . Phosphatidylserine is present on the surface of extracellular vesicles.
  • Extracellular vesicles can be created that display cytokines or target factors.
  • a PNE-tagged cytokine or target factor may be added to an extracellular vesicle in which a peptide neoepitope (PNE) nanobody has been expressed in advance and presented on the membrane surface of the extracellular vesicle.
  • PNE peptide neoepitope
  • a biotinylated cytokine or target factor may be added to the extracellular vesicle expressing streptavidin and displayed on the membrane surface of the extracellular vesicle.
  • a protein containing XXX and capable of presenting XXX to the outside of the membrane refers to a protein that contains at least XXX and is capable of presenting XXX to the outside of the membrane of an extracellular vesicle.
  • Protein capable of displaying XXX outside the membrane means that XXX and a membrane protein or a fragment containing a membrane-spanning domain thereof are combined using a plasmid or the like so that XXX is expressed on the membrane of cells or extracellular vesicles. It may also be expressed as a fusion protein containing.
  • a protein containing XXX and capable of presenting XXX to the outside of the membrane refers to soluble XXX (including, but not limited to, XXX itself; a fusion protein of XXX and the Fc portion of an antibody; and an antibody that recognizes XXX, or an antigen-binding fragment thereof (e.g., scFv, Fab, or nanobody), etc.), soluble XXX and extracellular vesicles are added as necessary. It may be bound to the membrane of an extracellular vesicle via a lipid linker, a peptide linker, or the like (for example, the method described in JP-A-2018-104341 may be referred to).
  • a desired tag for example, His tag, FLAG tag, PNE tag
  • soluble XXX for example, the tag is expressed as a fusion protein together with other components.
  • an antibody against the tag or its antigen-binding fragment for example, scFv, Fab, or nanobody, etc.
  • the protein containing e.g., an antibody against the tag or an antigen-binding fragment thereof (e.g., scFv, Fab, or nanobody) bound to the membrane of the extracellular vesicle via a linker or the like as necessary
  • the extracellular vesicle A membrane protein capable of being expressed in the membrane of the membrane protein or an extracellular vesicle containing in its membrane a nanobody for the tag (fusion protein, etc.) bound to the N-terminal side or C-terminal side of its transmembrane domain.
  • a PNE tag and an antibody against the tag as described in Raj D, et al., Gut., 2019 Jun;68(6):1052-1064, etc.
  • a PNE tag and an antibody against the tag as described in Raj D, et al., Gut., 2019 Jun;68(6):1052-1064, etc.
  • XXX formed by a multimer of subunits
  • one of the subunits is a protein that can be presented outside the membrane of an extracellular vesicle.
  • the remaining subunits do not need to be in a form that can be displayed outside the membrane.
  • one of the subunits is a protein that can be displayed outside the membrane of an extracellular vesicle, the other subunits can be displayed outside the membrane.
  • functional XXX can be assembled outside the membrane of extracellular vesicles.
  • membrane proteins or transmembrane domains thereof capable of being expressed on the membrane of extracellular vesicles include any protein that can be expressed on the membrane of extracellular vesicles. Any membrane protein or transmembrane domain thereof can be selected.
  • Membrane proteins or transmembrane domains thereof that can be expressed in the membrane of extracellular vesicles are membrane proteins that are known to be expressed in extracellular vesicles (e.g., exosomes, etc.) (e.g., tetraspanins). , CD58, ICAM-1, PTGFRN (for example, see Non-Patent Document 1, International Publication No. 2019/183578, etc.), or their transmembrane domains, etc. are preferable.
  • the "protein or domain thereof capable of binding to the membrane of extracellular vesicles” includes any protein as long as it is capable of binding to the membrane of extracellular vesicles. Or you can select that domain.
  • Proteins or domains thereof capable of binding to the membrane of extracellular vesicles are those known to be able to bind to the membrane of extracellular vesicles (for example, exosomes, etc.) (for example, MFG-E8 , or domains thereof (for example, the C1 and C2 domains of MFG-E8 described in Alain Delcayre, et al., Blood Cells, Molecules, and Diseases 35 (2005) 158-168), etc. are preferred.
  • the "membrane protein or its transmembrane domain capable of being expressed on the membrane of extracellular vesicles” or “the protein or its domain capable of binding to the membrane of extracellular vesicles” described herein is , may be from any animal species.
  • rodents such as mice and rats; lagomorphs such as rabbits; ungulates such as pigs, cows, goats, horses, and sheep; felines such as dogs and cats; humans, monkeys, rhesus monkeys, cynomolgus monkeys, marmosets, Examples include those derived from animals such as mammals such as primates such as orangutans and chimpanzees.
  • membrane protein or its transmembrane domain capable of being expressed on the membrane of extracellular vesicles or "the protein or its domain capable of binding to the membrane of extracellular vesicles” described herein is , preferably from a rodent or a mammal, more preferably from a mouse or a human.
  • mammalian extracellular vesicle markers are classified as follows.
  • Membrane proteins or GPI-anchored proteins that can be used as extracellular vesicle marker proteins include: 1) Non-tissue specific Tetraspanins (CD63, CD9, CD81, CD82), other multi-transmembrane membrane proteins (CD47, heterotrimeric G protein (GNA: Guanine nucleotide-binding proteins), etc.) MHC class I (HLA-A/B/C, H2-K/D/Q), Integrin (ITGA/ITGB), transferrin receptor (TFR2); LAMP1/2; Heparan sulfate proteoglycans (including syndecan (SDC)); Extracellular matrix metalloproteinase inducer (EMMPRIN) (also known as BSG or CD147); ADAM10; CD73 (NT5E), a GPI-anchored 5'nucleotidase; GPI-anchored complement fixation proteins CD55 and CD59; Sonic
  • proteins that are markers of extracellular vesicles are defined as "membrane proteins capable of being expressed on the membrane of extracellular vesicles” or “membrane proteins that bind to the membrane of extracellular vesicles” in the present invention, but are not limited thereto. It may also be used as a protein capable of
  • Tetraspanin as used herein means a protein belonging to the tetraspanin family (eg, but not limited to, CD9, CD53, CD63, CD81, CD82, CD151, etc.). Tetraspanins usually have, from the N-terminus, transmembrane domain 1 (hereinafter also referred to as "TM1”), small extracellular loop (hereinafter also referred to as "SEL”), and transmembrane domain 2 (hereinafter referred to as "TM2").
  • TM1 transmembrane domain 1
  • SEL small extracellular loop
  • TM2 transmembrane domain 2
  • TM3 small intracellular loop
  • LEL large extracellular loop
  • TM4 transmembrane domain 4
  • the amino acid sequence when the tetraspanin is mouse CD63, the amino acid sequence generally ranges from about 1 to about 110 and includes TM1, SEL, TM2, SIL, and TM3, and the amino acid sequence ranges from about 111 to about 200 and includes LEL, and the amino acid sequence ranges from about 201 to about 200. At about 238, it may contain TM4.
  • Each domain (for example, TM1, SEL, SIL, LTL, etc.) in the "tetraspanin" described herein may be derived from the same tetraspanin, or may be derived from a different tetraspanin in whole or in part. It may be.
  • the tetraspanin described herein has an amino acid sequence identity of 80% or more, preferably 80% or more, to its wild-type amino acid sequence, as long as it can be expressed in the membrane of extracellular vesicles. It may be 90% or more, more preferably 95% or more, still more preferably 98% or more, even more preferably 99% or more.
  • the tetraspanin described herein may contain a deletion of one or more amino acids from its wild-type amino acid sequence, as long as it can be expressed in the membrane of extracellular vesicles. , insertion, addition, and/or substitution.
  • the partial sequence of tetraspanin described herein (e.g., each domain; partial sequence including TM1, SEL, TM2, SIL, and TM3; partial sequence including TM4) has amino acid
  • the sequence identity may be 80% or more, preferably 90% or more, more preferably 95% or more, still more preferably 98% or more, even more preferably 99% or more.
  • the partial sequence of tetraspanin described herein is one in which one or more amino acids are deleted, inserted, added, and/or substituted with respect to its wild-type amino acid sequence. Good too.
  • MFG-E8 described herein has an amino acid sequence identity of 80% or more with respect to its wild type amino acid sequence, as long as it is capable of binding to the membrane of extracellular vesicles. Preferably it is 90% or more, more preferably 95% or more, still more preferably 98% or more, even more preferably 99% or more.
  • the MFG-E8 described herein may have one or more amino acids added to its wild-type amino acid sequence, as long as it is capable of binding to the membrane of extracellular vesicles. It may be deleted, inserted, added, and/or substituted.
  • CD58, PTGFRN, etc. described herein are capable of being expressed on the membrane of extracellular vesicles or capable of binding to the membrane of extracellular vesicles.
  • the amino acid sequence has an identity of 80% or more, preferably 90% or more, more preferably 95% or more, still more preferably 98% or more, even more preferably 99% or more with respect to the wild-type amino acid sequence. There may be.
  • CD58, PTGFRN, etc. described herein are capable of being expressed on the membrane of extracellular vesicles or capable of binding to the membrane of extracellular vesicles.
  • the wild-type amino acid sequence is one or more amino acids may be deleted, inserted, added, and/or substituted.
  • Spacer sequence refers to at least one protein that exists between two or more proteins or their subsequences or domains, etc. Refers to any sequence having amino acid residues.
  • a spacer sequence can be used, for example, when connecting two or more proteins or partial sequences or domains thereof. Spacer sequences are usually 1 to about 50 amino acid residues in length, preferably about 2 to about 28, and more preferably about 4 to about 25. Examples of spacer sequences include, but are not limited to, (GGGXS) n G m (wherein, each occurrence of X is independently A or G, and n is 1 to 8).
  • Polynucleotide as used herein means a single-stranded or double-stranded DNA molecule, an RNA molecule, a DNA-RNA chimera molecule, etc. Polynucleotides include genomic DNA, cDNA, hnRNA, mRNA, etc., and all naturally occurring or artificially modified derivatives thereof. Polynucleotides may be linear or circular.
  • At least one (1, 2, 3, 4 or 5) cytokines to identify each cytokine, hereinafter a first cytokine, a second cytokine, a further
  • the present invention provides extracellular vesicles that display cytokines (sometimes referred to as cytokines, etc.) outside the membrane.
  • at least one (1, 2, 3, 4 or 5) target factors hereinafter referred to as a first target factor, a second target factor, etc. to identify each target factor
  • the present invention provides extracellular vesicles that display extracellular vesicles (sometimes referred to as target factors, etc.) extracellularly.
  • at least one (1, 2, 3, 4 or 5) cytokines and at least one (1, 2, 3, 4 or 5) target factors are presented extramembranely. Provide extracellular vesicles.
  • an extracellular vesicle extramembranically displaying at least one cytokine the membrane comprising: (A) a protein that includes at least one cytokine or a subunit thereof and is capable of presenting the cytokine extramembrane; Provides extracellular vesicles containing.
  • an extracellular vesicle exhibiting at least one target factor extracellularly the membrane comprising: (B) a protein that includes at least one target factor or a subunit thereof and is capable of presenting the target factor outside the membrane; Provides extracellular vesicles containing.
  • an extracellular vesicle exhibiting at least one cytokine and at least one target factor extracellularly the membrane comprising: (A) A protein comprising at least one cytokine or a subunit thereof and capable of presenting the cytokine extramembrane; and (B) A protein comprising at least one target factor or a subunit thereof and capable of presenting the target factor outside the membrane. protein; Provides extracellular vesicles containing.
  • extracellular vesicles display cytokines and target factors extramembrane, the membrane comprising: (C) An extracellular vesicle is provided, which contains a first cytokine or a subunit thereof and a target factor, and includes a protein capable of displaying the first cytokine and the target factor extramembranely.
  • an extracellular vesicle extramembrane ly presents a first cytokine, a first target factor, and a second target factor
  • the membrane comprising: (A) a protein capable of displaying the first cytokine or a subunit thereof outside the membrane; (B) a protein capable of presenting the target factor to the outside of the membrane, including a first target factor or a subunit thereof; and (D) a protein capable of presenting the target factor to the outside of the membrane, including a second target factor or a subunit thereof;
  • Extracellular vesicles containing presentable proteins are provided.
  • an extracellular vesicle that presents a cytokine, a first target factor, and a second target factor extracellularly, the membrane comprising: (C) a protein comprising a first cytokine or a subunit thereof and a target factor and capable of presenting the first cytokine and target factor outside the membrane; and (D) a second target factor or a subunit thereof;
  • An extracellular vesicle containing a protein capable of displaying the second target factor outside the membrane is provided.
  • an extracellular vesicle that presents a cytokine, a first target factor, and a second target factor extracellularly, the membrane comprising: (E) a first cytokine or a subunit thereof, a first target factor or a subunit thereof, and a second target factor or a subunit thereof, the first cytokine, the first target factor, and the second target factor; extracellular vesicles containing a protein capable of extramembranely displaying a target factor.
  • the above-mentioned (A) "protein containing a cytokine or its subunit and capable of displaying the cytokine outside the membrane” is any other protein or protein thereof, as long as it is a protein capable of displaying the cytokine outside the membrane of an extracellular vesicle. It may also include a domain etc.
  • the above (A) binds a cytokine or a subunit thereof to a membrane protein or its transmembrane domain capable of being expressed on the membrane of an extracellular vesicle, or to the membrane of an extracellular vesicle. It is a fusion protein or protein complex that is capable of displaying the antigen outside the membrane, and includes a protein or a domain thereof that is capable of displaying the antigen.
  • the above (A) is: (A) A fusion protein capable of presenting the first cytokine outside the membrane, the protein comprising a first cytokine or a subunit thereof and a partial sequence of a tetraspanin, the partial sequence of the tetraspanin comprising two transmembrane or (A) a first cytokine or a subunit thereof and an MFG- E8 or a domain thereof, and is a fusion protein capable of displaying the first cytokine outside the membrane.
  • the partial sequence of a tetraspanin has at least two transmembrane domains, and the first cytokine or a subunit thereof is located between the two transmembrane domains.
  • the partial sequence of tetraspanin includes at least tetraspanin TM1 and TM2, and the first cytokine or its subunit is located between TM1 and TM2
  • the partial sequence of tetraspanin is Examples include a case where the first cytokine or a subunit thereof is located between TM3 and TM4, and the first cytokine or a subunit thereof is located between TM3 and TM4.
  • the above (A) is 1.
  • the first cytokine is a protein that can display the first cytokine outside the membrane.
  • tetraspanins can be expressed in membranes even if their large extracellular loops (LELs) are replaced in whole or in part with different amino acid sequences. It has been reported. Therefore, the first cytokine of (A-3) or its subunit may be inserted in place of the LEL of the tetraspanin via a spacer sequence that may be present, or may be inserted into the LEL of the tetraspanin or its subunit. It may be inserted at any position in the partial sequence.
  • A-1) "partial sequence of a tetraspanin comprising transmembrane domain 1, small extracellular loop, transmembrane domain 2, small intracellular loop, and transmembrane domain 3" usually includes transmembrane domain 4 of tetraspanin. do not have.
  • A-1) “Tetraspanin partial sequence including transmembrane domain 1, small extracellular loop, transmembrane domain 2, small intracellular loop, and transmembrane domain 3” does not include the large extracellular loop or its partial sequence. It's okay to stay.
  • each of the transmembrane domain 1, small extracellular loop, transmembrane domain 2, small intracellular loop, and transmembrane domain 3 may be a sequence derived from a different tetraspanin, or All sequences may be derived from the same tetraspanin.
  • transmembrane domain 1, small extracellular loop, transmembrane domain 2, small intracellular loop, and transmembrane domain 3 are all sequences derived from the same tetraspanin.
  • the partial sequence of tetraspanin comprising transmembrane domain 1, small extracellular loop, transmembrane domain 2, small intracellular loop and transmembrane domain 3 in (A-1) is all CD9 origin, a partial sequence derived from CD63 or CD81.
  • the partial sequences of tetraspanins comprising transmembrane domain 1, small extracellular loop, transmembrane domain 2, small intracellular loop and transmembrane domain 3 of (B-1) are all CD63 or CD81. This is a partial sequence derived from
  • the "partial sequence of tetraspanin containing transmembrane domain 4" in (A-5) usually includes transmembrane domain 1, small extracellular loop, transmembrane domain 2, small intracellular loop, and transmembrane domain 3 of tetraspanin. do not have.
  • the "partial sequence of tetraspanin containing transmembrane domain 4" of (A-5) may include a large extracellular loop or a partial sequence thereof.
  • the transmembrane domain 4 in (A-5) may be a sequence derived from a tetraspanin different from that in (A-1), or may be the same sequence derived from a tetraspanin as in (A-1).
  • transmembrane domain 4 in (A-5) is the same tetraspanin-derived sequence as in (A-1).
  • the partial sequence of tetraspanin containing transmembrane domain 4 in (A-5) is a partial sequence derived from CD9, CD63, or CD81.
  • the partial sequence of tetraspanin containing transmembrane domain 4 (A-5) is a partial sequence derived from CD63 or CD81.
  • the "partial sequence of tetraspanin comprising transmembrane domain 1, small extracellular loop, transmembrane domain 2, small intracellular loop, and transmembrane domain 3" of (A-1) is derived from CD63. and (A-5) "partial sequence of tetraspanin containing transmembrane domain 4" is a partial sequence derived from CD63.
  • the "partial sequence of tetraspanin comprising transmembrane domain 1, small extracellular loop, transmembrane domain 2, small intracellular loop, and transmembrane domain 3" of (A-1) is derived from CD81.
  • (A-5) "partial sequence of tetraspanin containing transmembrane domain 4" is a partial sequence derived from CD81.
  • MFG-E8 in (A-5) above preferably has SEQ ID NO: 37, 63, 73, 83, etc., or has an amino acid sequence identity of 80% or more, preferably 90% or more, and more preferably is 95% or more, more preferably 98% or more, even more preferably 99% or more.
  • the N-terminus of tetraspanins resides inside extracellular vesicles. Therefore, so that the cytokine of (A-3) or its subunit is presented on the outside of the extracellular vesicle, the "sequence containing any transmembrane domain and tetraspanin" of (A-5) above is (A-5) is a fusion of an amino acid sequence encoding an odd-numbered (1, 3, 5) transmembrane domain and a tetraspanin sequence so that the N terminus is located outside the extracellular vesicle. It is preferable.
  • CD8 single transmembrane protein
  • CD81 single transmembrane protein
  • the extracellular vesicles described herein may further contain a second (or more) cytokine in addition to the first cytokine.
  • the extracellular vesicles described herein may further comprise a second (or more) cytokine.
  • the second (or more) cytokine may be inserted into (A) above (for example, at the N-terminus and/or C-terminus of the "first cytokine" in (A-3)).
  • a second (or more) cytokine may be linked to the cytokine via a spacer sequence or the like as necessary).
  • the second (or more) cytokine has a structure similar to component (A) described herein, and thus is a protein of component (A) described herein (or Similar to the first cytokine, it may be included in the membrane of the antigen-presenting extracellular vesicles described herein as a separate protein (or fusion protein).
  • the "spacer sequence that may be present" in (A-2) and (A-4) of each of the above embodiments, if present, can be independently selected.
  • (A-2), if present, may be a spacer sequence such as SEQ ID NO: 31, 35, 45, 49, 61, 71, 81, 87, etc.
  • (A-4), if present, may be a spacer sequence such as SEQ ID NO: 31, 35, 45, 49, 61, 71, 81, 87, etc.
  • the cytokine when the cytokine has activity by forming a multimer (homo/heteromer) with multiple subunits, (A-6)
  • the extracellular vesicle may further contain subunits necessary for having activity.
  • a protein containing a target factor or a subunit thereof and capable of presenting the target factor to the outside of the membrane is a protein that can present the target factor to the outside of the membrane of an extracellular vesicle. It may also contain proteins or domains thereof.
  • the above (B) comprises a target factor or a subunit thereof, and a membrane protein or a transmembrane domain thereof capable of being expressed in the membrane of an extracellular vesicle, or A fusion protein or protein complex that includes a protein or domain thereof that is capable of binding and that is capable of displaying the antigen outside the membrane.
  • the above (B) comprises a target factor or a subunit thereof, and a membrane protein or a transmembrane domain thereof capable of being expressed in the membrane of an extracellular vesicle, or A fusion protein or protein complex that includes a protein or domain thereof that is capable of binding and that is capable of displaying the antigen outside the membrane.
  • the above (B) is: (B) A fusion protein capable of displaying the target factor outside the membrane, comprising a target factor or a subunit thereof, and a partial sequence of a tetraspanin, the partial sequence of the tetraspanin having at least two transmembrane domains.
  • a fusion protein capable of extramembrane display of the target factor comprising a target factor or a subunit thereof, and MFG-E8 or a domain thereof, or (B) a target factor or a subunit thereof, a tetraspanin, and optionally A fusion protein capable of extramembrane presentation of a target factor, containing any transmembrane domain that may be present.
  • the partial sequence of a tetraspanin has at least two transmembrane domains, and the first target factor or a subunit thereof is located between the two transmembrane domains
  • the partial sequence of tetraspanin includes at least TM1 and TM2 of tetraspanin, and the first target factor or its subunit is located between TM1 and TM2
  • the partial sequence of tetraspanin is For example, it includes at least the tetraspanins TM3 and TM4, and the first target factor or a subunit thereof is located between TM3 and TM4.
  • the above (B) is: 1.
  • B-1 A partial sequence of tetraspanin including, from the N-terminus, transmembrane domain 1, small extracellular loop, transmembrane domain 2, small intracellular loop, and transmembrane domain 3,
  • B-2) Spacer sequence that may be present,
  • B-3) Amino acid sequence of the first target factor or its subunit,
  • B-4) a spacer sequence that may be present; and
  • B-5) an amino acid sequence consisting of a partial sequence of a tetraspanin containing transmembrane domain 4, capable of presenting the first target factor outside the membrane.
  • fusion protein 2.
  • tetraspanins can be expressed in membranes even if their large extracellular loops (LELs) are replaced in whole or in part with different amino acid sequences. It has been reported. Therefore, the first target factor (B-3) or its subunit may be inserted in place of the LEL of the tetraspanin via a spacer sequence that may be present, or in the LEL of the tetraspanin or It may be inserted at any position within the partial sequence.
  • the "partial sequence of a tetraspanin comprising transmembrane domain 1, small extracellular loop, transmembrane domain 2, small intracellular loop, and transmembrane domain 3" in (B-1) usually includes transmembrane domain 4 of tetraspanin. do not have.
  • (B-1) “Tetraspanin partial sequence including transmembrane domain 1, small extracellular loop, transmembrane domain 2, small intracellular loop, and transmembrane domain 3” does not include the large extracellular loop or its partial sequence. It's okay to stay.
  • each of the transmembrane domain 1, small extracellular loop, transmembrane domain 2, small intracellular loop, and transmembrane domain 3 may be a sequence derived from a different tetraspanin, or All sequences may be derived from the same tetraspanin.
  • transmembrane domain 1, small extracellular loop, transmembrane domain 2, small intracellular loop, and transmembrane domain 3 are all sequences derived from the same tetraspanin.
  • the partial sequence of tetraspanin comprising transmembrane domain 1, small extracellular loop, transmembrane domain 2, small intracellular loop and transmembrane domain 3 in (B-1) is all CD9 origin, a partial sequence derived from CD63 or CD81.
  • the partial sequences of tetraspanins comprising transmembrane domain 1, small extracellular loop, transmembrane domain 2, small intracellular loop and transmembrane domain 3 of (B-1) are all CD63 or CD81. (subsequence derived from the origin).
  • transmembrane domain 4 “Tetraspanin partial sequence containing transmembrane domain 4” usually includes transmembrane domain 1, small extracellular loop, transmembrane domain 2, small intracellular loop, and transmembrane domain 3 of tetraspanin. do not have.
  • the "partial sequence of tetraspanin containing transmembrane domain 4" of (B-5) may include a large extracellular loop or a partial sequence thereof.
  • the transmembrane domain 4 in (B-5) may be a sequence derived from a tetraspanin different from that in (B-1), or may be the same sequence derived from a tetraspanin as in (B-1).
  • transmembrane domain 4 in (B-5) is the same tetraspanin-derived sequence as in (B-1).
  • the partial sequence of tetraspanin containing transmembrane domain 4 in (B-5) is a partial sequence derived from CD9, CD63, or CD81.
  • the partial sequence of tetraspanin containing transmembrane domain 4 in (B-5) is a partial sequence derived from CD63 or CD81.
  • the "partial sequence of tetraspanin comprising transmembrane domain 1, small extracellular loop, transmembrane domain 2, small intracellular loop, and transmembrane domain 3" of (B-1) is derived from CD63.
  • (B-5) "partial sequence of tetraspanin containing transmembrane domain 4" is a partial sequence derived from CD63.
  • the "partial sequence of tetraspanin comprising transmembrane domain 1, small extracellular loop, transmembrane domain 2, small intracellular loop, and transmembrane domain 3" of (B-1) is derived from CD81.
  • (B-5) "partial sequence of tetraspanin containing transmembrane domain 4" is a partial sequence derived from CD81.
  • MFG-E8 in (B-5) above preferably has SEQ ID NO: 37, 63, 73, 83, etc., or has an amino acid sequence identity of 80% or more, preferably 90% or more, and more preferably is 95% or more, more preferably 98% or more, even more preferably 99% or more.
  • (B-5) is a fusion of an amino acid sequence encoding an odd-numbered (1, 3, 5) transmembrane domain and a tetraspanin sequence so that the N terminus is located outside the extracellular vesicle. It is preferable that there be.
  • CD8 single transmembrane protein; e.g. SEQ ID NO: 5 or 21, or having an amino acid sequence identity of 80% or more, preferably 90% or more, more preferably 95% or more) , more preferably 98% or more, even more preferably 99% or more
  • CD81 single transmembrane protein; e.g. SEQ ID NO: 5 or 21, or having an amino acid sequence identity of 80% or more, preferably 90% or more, more preferably 95% or more
  • the extracellular vesicles described herein may further contain a second (or more) target factor in addition to the first target factor. Accordingly, in one embodiment of the invention, the extracellular vesicles described herein may further comprise a second (or more) targeting factor.
  • the second (or more) target factor or subunit thereof may be inserted into (B) above (for example, the "first target factor or subunit thereof" in (B-3)).
  • a second (or more) target factor or a subunit thereof may be linked to the N-terminal side and/or C-terminal side of the target factor, if necessary, via a spacer sequence, etc.).
  • the second (or more) target factor or its subunit has the same configuration as the component (B) described herein, thereby satisfying the component (B) described herein. ) as a separate protein (or fusion protein) from the protein (or fusion protein) contained in the membrane of the antigen-presenting extracellular vesicles described herein, similar to the first target factor.
  • Good i.e. constituent requirement (D) ).
  • the "spacer sequence that may be present" in (B-2) and (B-4) of each of the above embodiments, if present, can be independently selected.
  • (B-2), if present, may be a spacer sequence such as SEQ ID NO: 31, 35, 45, 49, 61, 71, 81, 87, etc.
  • (B-4), if present, may be a spacer sequence such as SEQ ID NO: 31, 35, 45, 49, 61, 71, 81, 87, etc.
  • the target factor when the target factor forms a multimer (homo/heteromer) with multiple subunits and has activity, (B-6)
  • the extracellular vesicle may further contain subunits necessary for having activity.
  • (A) and (B) may be fused into one molecule.
  • a fusion molecule may be translated as a single protein molecule with or without a spacer sequence between (A) and (B), or the proteins (A) and (B) may be chemically cross-linked. may be fused into one molecule by being bonded (for example, disulfide bond between cysteine residues).
  • (A) and (B) above are elements for localizing the protein in extracellular vesicles, i.e., "a membrane protein capable of being expressed in the membrane of an extracellular vesicle or its transmembrane domain.” ” or “a protein or domain thereof capable of binding to the membrane of extracellular vesicles” may be functionally fused.
  • the above-mentioned (C) "protein containing a cytokine or its subunit and a target factor or its subunit and capable of displaying the cytokine and the target factor outside the membrane” refers to The protein may contain other proteins or domains thereof, as long as the protein can be presented to the protein.
  • the above (C) comprises a cytokine or a subunit thereof, a target factor or a subunit thereof, and a membrane protein or a transmembrane domain thereof that can be localized in the membrane of an extracellular vesicle.
  • it may contain a protein capable of binding to the membrane of extracellular vesicles or a membrane-binding domain thereof.
  • the membrane protein capable of localizing in the membrane of extracellular vesicles or the protein capable of binding to the membrane of extracellular vesicles is a tetraspanin or MFG-E8; Good too.
  • the fusion protein includes, from the N-terminal side, (C-3) Amino acid sequence of target factor or its subunit, (C-4) a spacer sequence that may be present; and (C-5) a fusion peptide comprising a tetraspanin or its transmembrane domain or MFG-E8 or its transmembrane domain and the at least one cytokine. It may also include amino acid sequences encoding sequences.
  • the fusion protein includes, from the N-terminal side, (C-1) A fusion peptide comprising tetraspanin or its transmembrane domain or MFG-E8 or its transmembrane domain and the at least one cytokine (C-2) a spacer sequence that may be present, and (C- 3)
  • the amino acid sequence of the target factor or its subunit It may also contain amino acid sequences encoded in this order.
  • the fusion peptide includes, from the N-terminal side, (1) A partial sequence of a tetraspanin comprising transmembrane domain 1, small extracellular loop, transmembrane domain 2, small intracellular loop and transmembrane domain 3, (2) a spacer sequence that may be present; (3) the amino acid sequence of the at least one cytokine; (4) a spacer sequence that may be present; and (5) an amino acid sequence encoding a partial sequence of a tetraspanin containing transmembrane domain 4 in this order.
  • the fusion peptide includes, from the N-terminal side, (1) the amino acid sequence of at least one cytokine; (2) a spacer sequence that may be present; and (3) an amino acid sequence encoding the amino acid sequence of MFG-E8 or its membrane binding domain in this order.
  • the extracellular vesicle may further contain subunits necessary for having the activity.
  • the cytokine of each embodiment above is a T cell-stimulating cytokine.
  • the T cell stimulatory cytokine is IL-2 (preferably SEQ ID NO: 89, or with an amino acid sequence identity of 80% or more, preferably 90% or more, more preferably 95% or more) % or more, more preferably 98% or more, even more preferably 99% or more), IL-4, TGF- ⁇ , IL-7 (preferably SEQ ID NO: 93, or amino acid sequence identity thereto).
  • IL-15 preferably SEQ ID NO: 121, or amino acid sequence identity of 80% or more, preferably 90% or more, more preferably 95% or more, still more preferably 98% or more, even more preferably 99% or more.
  • those that can form multimers of homo or hetero subunits are functional (i.e., as long as they can have the desired pharmacological activity).
  • IL-12 preferably, TGF- ⁇ , etc.
  • IL-15 preferably SEQ ID NO: 121, or amino acid sequence identity of 80% or more, preferably 90% or more, more preferably 95% or more, still more preferably 98% or more, even more preferably 99% or more.
  • those that can form multimers of homo or hetero subunits for example, IL-12, TGF- ⁇ , etc.
  • are functional i.e., as long as they can have the desired pharmacological activity.
  • the Sushi domain of the IL-15 receptor (preferably SEQ ID NO: 117, or with which the amino acid sequence identity is 80% or more, preferably 90% or more, more preferably 95% or more, even more preferably (98% or more, more preferably 99% or more)) and may have a continuous amino acid sequence, optionally linked via a peptide linker or the like.
  • T cell-stimulating cytokines bind to the corresponding receptors present on the surface of cells such as T cells, B cells, and NK cells, and the signal is transmitted into the cells, thereby stimulating T cells, B cells, and NK cells. Differentiates and proliferates cells, monocytes, macrophages, etc.
  • the cytokine of each of the above embodiments is thrombopoietin (TPO; also referred to as Megakaryocyte Stimulating Factor) and/or stem cell factor (SCF; also referred to as Kit ligand).
  • TPO thrombopoietin
  • SCF stem cell factor
  • the first cytokine in each of the above embodiments is TPO (preferably SEQ ID NO: 19, or has an amino acid sequence identity of 80% or more, preferably 90% or more, more preferably SEQ ID NO: 19). is 95% or more, more preferably 98% or more, even more preferably 99% or more)
  • the second cytokine is SCF (preferably SEQ ID NO: 29 or 33, or an amino acid sequence identical to this).
  • the TPO gene encodes a protein of 353 amino acid residues, of which 21 residues are a signal sequence, and after removal, it is secreted as a 60-70 kDa glycoprotein.
  • TPO receptor By binding to c-mpl, a TPO receptor, and transmitting a signal into cells, it stimulates the proliferation of hematopoietic stem cells, the maturation and proliferation of megakaryocytes, and promotes the formation of platelets.
  • TPO also includes a fragment peptide of TPO having only a receptor binding domain.
  • the SCF gene encodes a 273-residue protein, and the N-terminal 25 residues are a signal sequence, and residues 26 to 273 are secreted as a membrane protein-type SCF that is sugar-modified, and its extracellular domain is processed. , released as soluble SCF.
  • SCF is a homodimer that binds to a receptor (two molecules) known as c-Kit (CD117) (heterotetramerization), and the KIT molecule is autophosphorylated and a signal is transmitted into the cell, thereby promoting hematopoietic stem cells. Propagation and maintenance, etc.
  • SCF includes membrane protein type, soluble type, and homodimer thereof bound with a linker.
  • the cytokine of each of the above embodiments is Activin or a subunit thereof.
  • Actinbin A is a product of the inhibitor ⁇ A chain preproprotein (also referred to as inhibitorn ⁇ A subunit precursor) gene product, which is cleaved by a processing enzyme and its C-terminal peptide ⁇ A subunit is homodimerized by SS bonds; actinbin B, in which the C-terminal peptide ⁇ B subunit, which is cleaved from the gene product by a processing enzyme (inhibin ⁇ B subunit precursor), is homodimerized by SS bonds; ⁇ A subunit and ⁇ B subunit are homodimerized by SS bonds; There is a heterodimerized actinbin AB.
  • the cytokine is the inhibitor ⁇ A chain preproprotein gene product itself or a fragment thereof (e.g., SEQ ID NO: 59, or 80% or more, preferably 90% or more, more preferably 95% or more, even more preferably 98% or more, even more preferably 99% or more of amino acid sequence identity to No. 69, or those having an amino acid sequence identity of 80% or more, preferably 90% or more, more preferably 95% or more, even more preferably 98% or more, even more preferably 99% or more) good.
  • SEQ ID NO: 59 e.g., SEQ ID NO: 59, or 80% or more, preferably 90% or more, more preferably 95% or more, even more preferably 98% or more, even more preferably 99% or more
  • These can function as active actinbin A within cells.
  • Activin A specifically binds to two forms of activin type I receptor (RI-A and RI-B) and two forms of activin type II receptor (RII-A and RII-B) to send signals into cells. is transmitted to induce differentiation of iPSCs (induced pluripotent stem cells)/ESCs (embryonic stem cells).
  • iPSCs induced pluripotent stem cells
  • ESCs embryonic stem cells
  • the target factor in each of the above embodiments is an antigen.
  • the targeting agent is an antigenic peptide.
  • the targeting factor is HER2 or a fragment thereof (preferably SEQ ID NO: 77 or 97, or with which the amino acid sequence identity is 80% or more, preferably 90% or more, more preferably 95%). % or more, more preferably 98% or more, even more preferably 99% or more), or CD19 or a fragment thereof.
  • antigenic peptides those that can form a multimer of homo or hetero subunits may be linked via a peptide linker, etc., as long as they are functional (that is, as long as they have the desired pharmacological activity).
  • it may be a continuous amino acid sequence.
  • the target factor in each of the above embodiments is a costimulatory molecule.
  • the targeting agent is a T cell costimulatory molecule.
  • the first target agent is an antigen and the second target agent is a T cell costimulatory molecule.
  • the target factor in each of the above embodiments is L-selectin and/or CXCL12.
  • the target factor is L-selectin (also referred to as CD62L) (preferably SEQ ID NO: 11, or has an amino acid sequence identity of 80% or more, preferably 90% or more, more preferably is 95% or more, more preferably 98% or more, even more preferably 99% or more) or CXCL12 (also referred to as SDF1) (preferably SEQ ID NO: 3, or has an amino acid sequence identity of 80% or more to this) , preferably 90% or more, more preferably 95% or more, still more preferably 98% or more, even more preferably 99% or more).
  • the first target factor may be L-selectin
  • the second target factor may be CXCL12.
  • L-selectin recognizes CD34 on hematopoietic stem cells (CD34 positive)
  • CXCL12 recognizes chemokine (C-X-C motif) receptor 4.
  • the target factor in each of the above embodiments is lecithin or a subunit thereof.
  • the lectin (sugar chain binding protein) Bc2Lc of Burkholderia cenocepacia bacteria is used.
  • the targeting factor is the Bc2Lc gene product or a multimer thereof linked via a linker (e.g. SEQ ID NOs: 43, 47, 51; may be 90% or more, more preferably 95% or more, still more preferably 98% or more, even more preferably 99% or more; or a multimer thereof).
  • Bc2Lc is a multimer that recognizes glycosylated proteins (eg, podocalyxin with Fuc ⁇ 1-2Gal ⁇ 1-3GalNAc) on the surface of iPSCs (induced pluripotent stem cells)/ESCs (embryonic stem cells).
  • glycosylated proteins eg, podocalyxin with Fuc ⁇ 1-2Gal ⁇ 1-3GalNAc
  • the extracellular vesicle is an exosome.
  • the extracellular vesicles described herein may contain or be bound to substances that may be therapeutically useful (e.g., low molecular weight compounds, nucleic acids, etc.) within or in their membranes. You can.
  • Methods for encapsulating the substance within the membrane of extracellular vesicles include, but are not limited to, mixing the substance and the extracellular vesicles described herein in a suitable solvent. Examples include a method to do so.
  • the extracellular vesicles may include any protein formulation.
  • Protein preparations include, but are not limited to, naturally occurring proteins such as erythropoietin, synthetic proteins that do not exist naturally such as immunoglobulin-CTLA4 fusion protein, and monoclonal antibodies or active fragments thereof. But that's fine. These protein preparations include membrane proteins capable of localizing in the membrane of extracellular vesicles or fusion proteins with their transmembrane domains or proteins capable of binding to the membrane of extracellular vesicles or their membrane-binding domains. It may also be localized on the surface of extracellular vesicles. Such extracellular vesicles can be obtained by transfecting cells that produce extracellular vesicles with a vector for expressing the fusion protein, and causing the cells to secrete the extracellular vesicles.
  • Each fusion protein or protein complex or protein formulation contained in the membrane of the extracellular vesicles described herein may include one or more detectable labels.
  • a fusion protein or protein complex or protein preparation may be labeled with a specific reporter molecule, fluorophore, radioactive material, enzyme (eg, peroxidase, phosphatase), etc. in a conventional manner. These may be linked to the N-terminus or C-terminus of the fusion protein, protein complex, or protein preparation, for example, as a component of the fusion protein, protein complex, or protein preparation.
  • each protein (or fusion protein) in (A), (B), (C), (D) and (E) contained in the membrane of the extracellular vesicle described herein ) is provided.
  • sequence encoding a fusion protein (D) comprising a second targeting factor or a subunit thereof and capable of presenting a targeting factor outside the membrane of an extracellular vesicle; and (e) at least one cytokine or its subunits; a subunit, a first target factor or a subunit thereof, and a second target factor or a subunit thereof;
  • Sequence encoding a fusion protein (E) that can be displayed externally
  • a polynucleotide comprising at least one sequence selected from the group consisting of:
  • the above sequences (a) to (e) are highly homologous to the sequences specifically described in the present specification (preferably 90% or more, more preferably 95% or more, still more preferably 99% or more homologous) gender), but is not particularly limited. They may be paralogs (gene sequences resulting from gene duplication) or orthologs (groups of genes with homologous functions existing in different organisms) as long as they have equivalent functions; This also includes
  • each protein (or fusion protein) in (A) to (E) is the amino acid sequence of each component in each fusion protein or protein complex (for example, in the case of (A), (A-1) It can be appropriately determined with reference to the amino acid sequences of ⁇ (A-5) or (A-3) ⁇ (A-5), and optionally (A-6)).
  • Any type of codon can be selected to be used when determining a polynucleotide.
  • the polynucleotide may be determined in consideration of the codon frequency of cells to be transformed using a vector containing the polynucleotide.
  • a polynucleotide encoding a signal peptide may be added to the N-terminus of the polynucleotide encoding each protein (or fusion protein) in (A) to (E) above. .
  • any amino acid sequence of the signal peptide can be used, and may be determined by taking into consideration, for example, the amino acid sequence of the fusion protein to be expressed.
  • polynucleotides encoding signal peptides include polynucleotides encoding CD8 signal peptides (eg, SEQ ID NOs: 1, 17, and 41) (eg, SEQ ID NOs: 2, 18, and 42), and MEG-E8 signal peptides. (eg, SEQ ID NO: 27) (eg, SEQ ID NO: 28), and the like.
  • each component of each protein (or fusion protein) in (A) to (E) described above (for example, in the case of (A), (A-1) to (A-5) or (A-3) ⁇ (A-5), optionally (A-6)), signal peptides, and other amino acid sequences, as well as information on polynucleotides encoding these, can be found, for example, in known documents or from NCBI (http://www.ncbi You may also search a database such as .nlm.nih.gov/guide/) and obtain it as appropriate. Furthermore, for the amino acid sequence in the partial sequence of tetraspanin and the polynucleotide encoding the same, International Publication No. 2016/139354 may be referred to.
  • One embodiment of the present invention provides a vector comprising at least one polynucleotide selected from the polynucleotides described herein.
  • Vector refers to any vector, including, but not limited to, plasmid vectors, cosmid vectors, phage vectors such as phages, adenovirus vectors, baculovirus vectors, etc. (including viral vectors, artificial chromosome vectors, etc.).
  • Vectors include expression vectors, cloning vectors, and the like.
  • Expression vectors generally contain the desired coding sequence and appropriate polynucleotides necessary for expression of the operably linked coding sequence in a host organism (e.g., plant, insect, animal, etc.) or in vitro expression system. may contain.
  • Cloning vectors may be used to manipulate and/or amplify desired polynucleotide fragments. Cloning vectors may lack functional sequences required for expression of the desired polynucleotide fragment.
  • the polynucleotides described herein may all be inserted into the same vector, as long as they can be operably inserted, or two or more polynucleotides may be inserted into separate vectors. It may be inserted into a vector.
  • a kit is provided that combines two or more vectors comprising at least one polynucleotide selected from the polynucleotides described herein.
  • Transformed Cells In one embodiment of the invention: (i) a polynucleotide encoding at least one protein (or fusion protein) of (A) as described herein; (ii) a polynucleotide encoding at least one protein (or fusion protein) of (B) as described herein; (iii) a polynucleotide encoding at least one protein (or fusion protein) of (C) as described herein; (iv) a polynucleotide encoding at least one (D) protein (or fusion protein) as described herein; and/or (v) at least one (E) protein as described herein. (or a fusion protein) that has been transformed with a single vector or a combination of two or more vectors.
  • At least one polynucleotide of (a) as described herein at least one polynucleotide of (b) as described herein, and/or (iii) at least one polynucleotide of (c) as described herein; and/or (v) a polynucleotide encoding a protein (or fusion protein) of at least one (e) polynucleotide as described herein.
  • Cells transformed with one vector or a combination of two or more vectors are provided.
  • Transformed with a single vector or a combination of two or more vectors means, for example, that cells are transformed with polynucleotides (i) to (v) above all inserted into the same vector. It means that they may be transformed or may be transformed by a combination of two or more vectors, two or more of which are inserted into separate vectors.
  • the cells to be transformed are not particularly limited as long as the extracellular vesicles described herein can be obtained after transformation, and even if they are primary cultured cells, subcultured cells or established cell lines, and these may be normal cells or diseased cells including cancerous or tumorous cells.
  • the origin of cells to be transformed is not particularly limited, but examples include rodents such as mice, rats, hamsters, and guinea pigs, lagomorphs such as rabbits, pigs, cows, goats, horses, sheep, etc.
  • Cells derived from animals such as ungulates, dogs, cats, and other mammals; humans, monkeys, rhesus monkeys, cynomolgus monkeys, marmosets, orangutans, and primates such as chimpanzees; cells derived from plants; cells derived from insects. etc.
  • the cells to be transformed are preferably cells derived from animals.
  • animal-derived cells include, but are not limited to, human embryonic kidney cells (including HEK293T cells, etc.), human FL cells, Chinese hamster ovary cells (CHO cells), COS-7, Vero, Examples include mouse L cells and rat GH3.
  • the method for transforming cells is not particularly limited as long as it can introduce the polynucleotide of interest into the cells.
  • electroporation method microinjection method, calcium phosphate method, cationic lipid method, method using liposome, method using non-liposomal material such as polyethyleneimine, virus infection method, etc. may be used.
  • the transformed cell may be a transformed cell that transiently expresses the protein (or fusion protein) of (A), (B), (C), (D) and/or (E). Alternatively, it may be a transformed cell (stable cell line) that stably expresses it.
  • the culture conditions for transformed cells are not particularly limited.
  • the transformed cell is an animal-derived cell, for example, a medium commonly used for cell culture, etc. (e.g., RPMI1640 medium, Eagle's MEM medium, Dulbecco's modified Eagle medium (DMEM medium), Ham F12
  • a medium commonly used for cell culture, etc. e.g., RPMI1640 medium, Eagle's MEM medium, Dulbecco's modified Eagle medium (DMEM medium), Ham F12
  • a desired period of time e.g., about 0.5 to about 240 hours (preferably about 5 to about 120 hours). , more preferably for about 12 to about 72 hours
  • cultured for example, under standing or shaking.
  • the culture supernatant obtained by culturing transformed cells may contain the extracellular vesicles described herein. Therefore, when culturing transformed cells for the purpose of obtaining the antigen-presenting extracellular vesicles described herein, if necessary, a medium from which extracellular vesicles such as exosomes have been removed (for example, a medium from which exosomes have been removed) Dulbecco's modified Eagle's medium containing about 1 to about 5% fetal bovine serum, etc.) may also be used.
  • a medium from which extracellular vesicles such as exosomes have been removed for example, a medium from which exosomes have been removed
  • Dulbecco's modified Eagle's medium containing about 1 to about 5% fetal bovine serum, etc.
  • a culture supernatant obtained by culturing the transformed cells described herein is provided.
  • Extracellular vesicles contained in the culture supernatant described herein can be recovered by, for example, purifying (e.g., centrifugation, chromatography, etc.), concentrating, isolating, etc. the culture supernatant. can do.
  • extracellular vesicles obtained from the culture supernatants described herein are provided.
  • the extracellular vesicles described herein can be produced, for example, by means such as, but not limited to, genetic recombination techniques known to those skilled in the art (e.g., by the methods described below or in the Examples). or a method similar thereto.
  • each may be operably linked to the same or different promoters.
  • the obtained single or two or more vectors are transformed into cells simultaneously or sequentially, and the transformed cells (which may be transformed cells that express these fusion proteins transiently or stably) It is possible to obtain transformed cells (which may be stable strains) that express the same expression.
  • the obtained transformed cells are cultured under desired conditions to obtain a culture supernatant, and the obtained culture supernatant is purified as necessary (e.g. centrifugation, antibody (e.g. extracellular vesicle membrane)
  • the extracellular vesicles described herein can be purified by purification using an antibody that recognizes the protein contained therein), chromatography, flow cytometry, etc., concentration (e.g., ultrafiltration, etc.), drying, etc. can be obtained.
  • the extracellular vesicles described herein can be detected by methods such as flow cytometry, ELISA, and Western blotting. It may be confirmed that (C) instead of; (A) and (B) and (E) instead of (D) are included in the film.
  • a method for producing an antigen-presenting extracellular vesicle as described herein comprising: A method is provided, comprising collecting a culture supernatant.
  • a method for producing an extracellular vesicle as described herein comprising: (i) a polynucleotide (or polynucleotide (a)) encoding the protein (A) (or fusion protein) described herein; (ii) a polynucleotide (or polynucleotide (b)) encoding the protein (B) (or fusion protein) described herein; (iii) a polynucleotide (or polynucleotide (c)) encoding the protein (C) (or fusion protein) described herein; (iv) a polynucleotide (or polynucleotide of (d)) encoding the protein (D) (or fusion protein) described herein, and/or (v) (E) as described herein; ) (or polynucleotide of (e)) encoding the protein (or fusion protein) transforming cells simultaneously or sequentially (preferably
  • a composition comprising an antigen-presenting extracellular vesicle, a polynucleotide and/or a vector comprising the same, and/or a transformed cell and/or a culture supernatant thereof, as described herein.
  • a pharmaceutical composition e.g., a pharmaceutical composition.
  • medicaments and reagents are provided that include antigen-presenting extracellular vesicles as described herein, or culture supernatants as described herein.
  • compositions described herein include, but are not limited to, excipients, lubricants, binders, disintegrants, pH adjusters, Additives such as solvents, solubilizers, suspending agents, tonicity agents, buffers, soothing agents, preservatives, antioxidants, colorants, sweeteners, surfactants, and the like can be included.
  • additives such as solvents, solubilizers, suspending agents, tonicity agents, buffers, soothing agents, preservatives, antioxidants, colorants, sweeteners, surfactants, and the like can be included.
  • the type of these additives, the amount used, etc. can be appropriately selected by those skilled in the art depending on the purpose.
  • these additives are preferably pharmacologically acceptable carriers.
  • composition described herein contains a polynucleotide
  • a carrier suitable for DD (drug delivery) of the nucleic acid include lipid nanoparticles (LNPs). and polymers (eg PEI).
  • composition described herein can be prepared, for example, into tablets, coated tablets, orally disintegrating tablets, chewable preparations, pills, etc., together with the above-mentioned additives, by a method known per se.
  • the formulation may further contain other beneficial ingredients (eg, other therapeutically beneficial ingredients) depending on its purpose.
  • the composition that is an embodiment of the present invention can be used to generate genetically modified T cells that recognize the antigen, such as T cells that recognize and bind to the antigen.
  • T cells TCR-T cells in which the cell receptor (TCR) is forcibly expressed; the region that recognizes and binds to the antigen (e.g., single chain variable region scFv (scFv) containing VH and VL, and heavy chain A fusion protein containing a lymphocyte activation molecule (e.g., the intracellular domain of CD28 or the intracellular domain of mCD3z), such as a nanobody containing the variable region of an immunoglobulin (antibody) consisting of a target antigen recognition portion of an antibody (for example, the intracellular domain of CD28 or the intracellular domain of mCD3z).
  • scFv single chain variable region scFv
  • mCD3z heavy chain A fusion protein containing a lymphocyte activation molecule
  • CAR-T cells chimeric antigen receptor
  • CAR-T cells chimeric antigen receptor
  • the pharmaceutical composition that is an embodiment of the present invention to a subject, the T cell receptor of TCR-T cells, CAR -
  • the antigen presented on the extracellular membrane of the extracellular vesicle reacts with a chimeric antigen receptor, etc. of a T cell, and preferably also with a T cell-stimulating cytokine receptor on a TCR-T cell, a CAR-T cell, etc.
  • T cell costimulatory molecules are also presented outside the membrane of extracellular vesicles, they interact with molecules (e.g., CD28, CD134, etc.) present on the membrane of TCR-T cells, CAR-T cells, etc. This further contributes to the activation and/or proliferation of TCR-T cells, CAR-T cells, etc.
  • Activated and/or proliferated TCR-T cells, CAR-T cells, etc. attack cancer cells that express the antigen or its fragment on the surface, suppressing the proliferation of cancer cells and treating cancer. do.
  • Cancer includes any solid cancer and blood cancer, but is not limited to, for example, small cell lung cancer, non-small cell lung cancer, breast cancer, esophageal cancer, stomach cancer, small intestine cancer. , colorectal cancer, colon cancer, rectal cancer, pancreatic cancer, prostate cancer, bone marrow cancer, kidney cancer (including renal cell cancer, etc.), parathyroid cancer, adrenal gland cancer, and ureteral cancer.
  • cancer liver cancer, bile duct cancer, cervical cancer, ovarian cancer (for example, the histological types are serous adenocarcinoma, mucinous adenocarcinoma, clear cell adenocarcinoma, etc.), testicular cancer, and bladder cancer.
  • Vulvar cancer penile cancer, thyroid cancer, head and neck cancer, craniopharyngeal cancer, pharyngeal cancer, tongue cancer, skin cancer, Merkel cell cancer, melanoma (malignant melanoma, etc.) , epithelial cancer, squamous cell carcinoma, basal cell carcinoma, pediatric cancer, cancer of unknown primary origin, fibrosarcoma, mucosal sarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, lymphangiosarcoma , intralymphatic sarcoma, Kaposi's sarcoma, leiomyosarcoma, rhabdomyosarcoma, synovioma, mesothelioma, Ewing tumor, seminoma, Wilms tumor, brain tumor, glioma, glioblastoma, astrocytoma , medulloblastoma, men
  • the composition that is an embodiment of the present invention is capable of activating/proliferating endogenous or externally transplanted hematopoietic stem cells in vitro or in vivo. Therefore, it can be used for recovery treatment of hematopoietic stem cells against aplastic anemia and decrease in blood cells after cancer radiotherapy.
  • Aplastic anemia causes various symptoms due to a decrease in red blood cells, neutrophils, and platelets. Transplanting healthy hematopoietic stem cells to treat or prevent aplastic anemia by regenerating the patient's hematopoietic capacity.
  • Hematopoietic stem cell transplants are also performed for the purpose of completely curing blood cancers and immunodeficiency diseases that are difficult to cure with conventional chemotherapy and immunosuppressive therapy alone.
  • pre-transplant treatment which includes high-dose chemotherapy and whole-body radiation therapy
  • hematopoietic stem cells collected in advance from the patient or from a donor are administered via an intravenous drip.
  • Cancers that are easily treated with chemotherapy or radiation therapy, such as blood or lymph cancer are suitable for treatment.
  • the purpose of pre-transplant treatment is to reduce tumor cells and suppress the patient's own immune cells.
  • the transplanted hematopoietic stem cells take root (engraft) in the patient's bone marrow, restoring normal hematopoietic function.
  • allogeneic hematopoietic stem cell transplantation allogeneic transplantation
  • a graft-versus-leukemia effect in which the donor's lymphocytes attack the patient's tumor cells can be expected.
  • the composition that is an embodiment of the present invention is capable of inducing differentiation of iPS cells/embryonic stem cells in vitro or in vivo.
  • Subjects to be treated or prevented for the various diseases mentioned above include, but are not limited to, rodents such as mice, rats, hamsters, and guinea pigs; lagomorphs such as rabbits; pigs, and cows. , animals such as ungulates such as goats, horses, and sheep; felids such as dogs and cats; primates such as humans, monkeys, rhesus monkeys, cynomolgus monkeys, marmosets, orangutans, and chimpanzees; and plants such as mammals.
  • the animal is preferably an animal, more preferably a rodent or a primate, and even more preferably a mouse or a human.
  • the dosage of the drug can be determined as appropriate by taking into account the sex, age, weight, health condition, degree of medical condition, or diet of the subject to be administered; administration time; administration method; combination with other drugs; and other factors. can.
  • Example 1 Effect on hematopoietic stem cells 1.1. Preparation of plasmids A polynucleotide (SEQ ID NO: 2) encoding the signal peptide of CD8A (SEQ ID NO: 1), a polynucleotide (SEQ ID NO: 4) encoding the full-length sequence of CXCL12 (SEQ ID NO: 3), a polynucleotide encoding the signal peptide of CD8A (SEQ ID NO: 1), a polynucleotide (SEQ ID NO: 4) encoding the full-length sequence of CXCL12 (SEQ ID NO: 3), An artificially synthesized gene sequence (SEQ ID NO: 9) consisting of a polynucleotide (SEQ ID NO: 6) encoding the full-length sequence (SEQ ID NO: 5) and a polynucleotide (SEQ ID NO: 8) encoding the full-length sequence of CD81 (SEQ ID NO: 7).
  • pcDNA (trademark) 3.1 (+) Mammalian Expression Vector (manufactured by Thermo Fisher Scientific) to express CXCL-12 on the membrane of extracellular vesicles.
  • pcDNA (trademark) 3.1 (+) Mammalian Expression Vector (manufactured by Thermo Fisher Scientific) to express CXCL-12 on the membrane of extracellular vesicles.
  • pcDNA (trademark) 3.1 (+) Mammalian Expression Vector (manufactured by Thermo Fisher Scientific) to express CXCL-12 on the membrane of extracellular vesicles.
  • pcDNA (trademark) 3.1 (+) Mammalian Expression Vector (manufactured by Thermo Fisher Scientific) to express CXCL-12 on the membrane of extracellular vesicles.
  • SEQ ID NO: 12 a polynucleotide encoding the full-length sequence of L-Selectin
  • SEQ ID NO: 14 polynucleotide
  • the polynucleotide (SEQ ID NO: 16) encoding the artificially synthesized gene sequence (SEQ ID NO: 15) was added to pcDNA (trademark) 3.1 (+) Mammalian Expression Vector (manufactured by Thermo Fisher Scientific), and the signal peptide of CD8A (SEQ ID NO: 17) (SEQ ID NO: 18), a polynucleotide encoding the full-length sequence of Thrombopoietin (TPO) (SEQ ID NO: 19) (SEQ ID NO: 20), a full-length sequence of CD8A excluding the signal peptide (SEQ ID NO: 21)
  • a polynucleotide (SEQ ID NO: 22) encoding an artificially synthesized gene sequence (SEQ ID NO: 25) consisting of a polynucleotide (SEQ ID NO: 24) encoding the full-length sequence of CD81 (SEQ ID NO: 23) SEQ ID NO: 26) was added
  • HEK293 HEK293 cells derived from human embryonic kidney (manufactured by JCRB Cell Bank) were grown in E-MEM medium containing 10% FBS (L- Preculture was performed using glutamine (containing phenol red) (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.).
  • HEK293 was seeded at 1 ⁇ 10 6 cells/10 mL/10 cm dish and cultured in a static state in a CO 2 incubator (37° C., 5% CO 2 ) for 24 hours.
  • Example 1 Lipofectamine (registered trademark) 3000 Reagent (manufactured by Thermo Fisher Scientific) and Opti-MEM (trademark) I Reduced Serum Medium, no phenol red (Thermo Fisher Scientific) were added. isher Scientific) was used to test the non-vector-introduced group (sample 1) Introduce 5 ⁇ g each of SCF and TPO expression vector introduction group (sample 2), SCF, TPO and L-Selectin expression vector introduction group (sample 3), and SCF, TPO and CXCL12 expression vector introduction group (sample 4), The cells were cultured in a CO 2 incubator (37° C., 5% CO 2 ) for 24 hours.
  • a CO 2 incubator 37° C., 5% CO 2
  • the collected culture supernatant was concentrated using Vivaspin 20 (100k) (manufactured by Sartorius), and the concentrated solution was used to perform PS Capture (trademark) exosome flow cytometry kit (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.). Immunostaining was performed according to the manufacturer's instructions.
  • the antibodies used for staining are as follows. The staining time was 60 minutes for both the primary antibody and the secondary antibody at 4°C. After staining, the expression of each fusion protein was detected using a flow cytometer LSRFortessaX-20 (manufactured by BD Biosciences).
  • HEK293 cell-derived extracellular vesicles and quantitative evaluation of SCF and TPO Human fetal kidney-derived HEK293 cells (manufactured by JCRB Cell Bank) were grown in E-MEM medium containing 10% FBS (containing L-glutamine and phenol red) (Fujifilm Wako Pure Preculture was carried out using a commercially available product (manufactured by Yakusha).
  • HEK293 was seeded at 1 ⁇ 10 6 cells/10 mL/10 cm dish and cultured in a static state in a CO 2 incubator (37° C., 5% CO 2 ) for 24 hours.
  • Lipofectamine 3000 Reagent manufactured by Thermo Fisher Scientific
  • Opti-MEM I Reduced Serum Medium no phenol red (manufactured by Thermo Fisher Scientific) were added.
  • the vector-unintroduced group (sample 1), SCF and TPO expression 5 ⁇ g each of the vector introduction group (sample 2), the SCF, TPO and L-Selectin expression vector introduction group (sample 3), and the SCF, TPO and CXCL12 expression vector introduction group (sample 4) were introduced and placed in a CO 2 incubator (37°C, The cells were cultured for 24 hours in 5% CO 2 ).
  • each 10 cm dish washed with D-PBS was treated with E-MEM medium (L-glutamine, 10 mL of phenol red (containing phenol red) and cultured in a CO 2 incubator (37° C., 5% CO 2 ) for 48 hours. After 48 hours, the culture supernatant was collected. The collected culture supernatant was centrifuged at 2000xg for 10 minutes, the supernatant was collected, and then passed through a 0.22 ⁇ m filter (manufactured by Millipore).
  • E-MEM medium L-glutamine, 10 mL of phenol red (containing phenol red)
  • CO 2 incubator 37° C., 5% CO 2
  • the treated culture supernatant was added to a UC tube (manufactured by Beckman Coulter), set in SW41Ti (manufactured by Beckman Coulter), and incubated at 35,000 rpm using Optima L-90K (manufactured by Beckman Coulter). Centrifugation was performed at °C for 70 minutes. After centrifugation, the supernatant was removed, 10 mL of D-PBS was added to the UC tube, and the mixture was centrifuged at 35,000 rpm and 4°C for 70 minutes. After centrifugation, the supernatant was removed and suspended in 50 ⁇ L of D-PBS.
  • Human SCF ELISA Kit manufactured by ABCAM
  • Human Thrombopoietin ELISA Kit manufactured by ABCAM
  • 50 ⁇ L of a 10-fold diluted extracellular vesicle solution or a serially diluted standard curve solution was added to each plate used.
  • 50 ⁇ L of Antibody Cocktail prepared with Antibody Diluent CPI or Antibody Diluent 5BI to a final concentration of 1 ⁇ Capture Antibody and 1 ⁇ Detector Antibody, and shake at room temperature for 1 hour. Last night. After 1 hour, the solution was discarded and washed three times with 350 uL of 1 ⁇ Wash Buffer PT.
  • SCF and TPO The final concentrations of SCF and TPO are SCF: 3 pg/mL, TPO: 1.63 pg/mL (sample 2); SCF: 3 pg/mL, TPO: 3.69 pg/mL (sample 3) and SCF: 3 pg/mL, TPO :
  • Each extracellular vesicle solution prepared in 1.2.2 above was added so that the concentration was 2.92 pg/mL (sample 4).
  • Sample 1 was added in a manner that matched Sample 2, which had the largest amount added.
  • recombinant SCF manufactured by R&D Systems
  • recombinant TPO manufactured by Peprotech
  • a D-PBS treatment group was set as a control.
  • the final volume of each group was adjusted with the medium and D-PBS solution so that it was 100 ⁇ L.
  • the treated plates were cultured in a CO 2 incubator (37° C., 5% CO 2 ) for 96 hours.
  • a polynucleotide encoding an artificially synthesized gene sequence consisting of a polynucleotide (SEQ ID NO: 64) encoding the full-length sequence of MFGE8 excluding the signal peptide (SEQ ID NO: 63) (SEQ ID NO: 63); 66) into pcDNA (trademark) 3.1/Zeo(+) Mammalian Expression Vector (manufactured by Thermo Fisher Scientific), a polynucleotide (SEQ ID NO: 68) encoding the signal peptide of MFGE8 (SEQ ID NO: 67), and Mature Activin.
  • a polynucleotide (SEQ ID NO: 76) encoding an artificially synthesized gene sequence (SEQ ID NO: 75) consisting of a polynucleotide (SEQ ID NO: 74) encoding SEQ ID NO: 73) was added to pcDNA(TM) 3.1/Zeo(+ ) Mammalian Expression Vector (manufactured by Thermo Fisher Scientific) to prepare vectors for expressing Mutation Activin A and Mature Activin A on the membrane of extracellular vesicles.
  • Each constructed vector was E.
  • the cells were transformed into E. coli DH5 ⁇ Competent Cells (manufactured by Takara Bio Inc.).
  • the transformed E. coli was amplified using LB medium, and a large amount of vector was prepared using EndoFree Plasmid Maxi Kit (manufactured by QIAGEN).
  • the sequence information used is shown in Tables 6 to 8 below.
  • HEK293 cell-derived extracellular vesicles and flow cytometry analysis of fusion proteins contained in the membrane of the extracellular vesicles
  • Human embryonic kidney-derived HEK293 cells (manufactured by JCRB Cell Bank) were grown in E-MEM medium containing 10% FBS (L- Preculture was performed using glutamine (containing phenol red) (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.).
  • HEK293 was seeded at 8 ⁇ 10 5 cells/10 mL/10 cm dish and cultured in a static state in a CO 2 incubator (37° C., 5% CO 2 ) for 24 hours.
  • Immunostaining was performed using the concentrated solution using PS Capture (trademark) Exosome Flow Cytometry Kit (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) according to the manufacturer's instructions.
  • PS Capture trademark
  • AlexaFluor registered trademark
  • 647 conjugate anti-human Activin A antibody manufactured by Bioss antibodies
  • reaction was performed at room temperature for 60 minutes.
  • the expression of each fusion protein was detected using a flow cytometer LSRFortessaX-20 (manufactured by BD Biosciences).
  • HEK293 cell-derived extracellular vesicles and quantitative evaluation of Activin A Human fetal kidney-derived HEK293 cells (manufactured by JCRB Cell Bank) were cultured in E-MEM medium containing 10% FBS (containing L-glutamine and phenol red) (Fujifilm Wako Pure Chemical Industries, Ltd.) Preculture was carried out using a commercially available commercially available product. HEK293 was seeded at 8 ⁇ 10 5 cells/10 mL/10 cm dish and cultured in a static state in a CO 2 incubator (37° C., 5% CO 2 ) for 24 hours.
  • FBS containing L-glutamine and phenol red
  • Mutation Activin A expression vector introduction group (Example conditions 1) 7.5 ⁇ g of each vector, 3.75 ⁇ g of each vector for the Bc2l-C and Mutation Activin A expression vector introduced group (Example conditions 2), and 7.5 ⁇ g of each vector for the Mature Activin A expression vector introduced group (Example conditions 3). and cultured in a CO 2 incubator (37° C., 5% CO 2 ) for 24 hours.
  • a group without vector introduction that was operated in the same manner was used as a comparative example. After 24 hours, each 10 cm dish washed with D-PBS was treated with E-MEM medium (L-glutamine) containing 10% Fetal Bovine Serum, exosome-depleted, One Shot format (manufactured by Thermo Fisher Scientific). , 10 mL of phenol red (containing phenol red) and cultured in a CO 2 incubator (37° C., 5% CO 2 ) for 48 hours. After 48 hours, the culture supernatant was collected.
  • E-MEM medium L-glutamine
  • phenol red containing phenol red
  • CO 2 incubator 37° C., 5% CO 2
  • Extracellular vesicles from the collected culture supernatant were purified using Capturem (trademark) Extracellular Vesicle Isolation Kit (Maxi) (manufactured by Takara Bio Inc.), and the resulting solution was purified using Amicon Ultra-0.5, PLGC Ultracel, It was concentrated using 10kDa (manufactured by Merck Millipore) and replaced with D-PBS(-).
  • the concentrated solution was measured using Human Activin A ELISA (manufactured by RayBiotech).
  • the number and average particle diameter of extracellular vesicles contained in the same solution were measured using a nanoparticle tracking analyzer ZetaView (manufactured by DKSH Japan).
  • the obtained cells were suspended in StemFit (registered trademark) medium supplemented with 10 ⁇ M Y-27632 (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) and placed in a 24-well plate coated with iMatrix-511 at 0.5 ⁇ g/cm 2 .
  • the cells were seeded at 2 ⁇ 10 5 cells/well and cultured in a static state in a CO 2 incubator (37° C., 5% CO 2 ) for 24 hours. (Day-1). The day after seeding, it was confirmed that the cells had adhered, and the medium was replaced with a differentiation medium to start differentiation (Day 0).
  • the differentiation medium contained 2mM L-glutamine (manufactured by DS Pharma Biomedical), 1% MEM non-essential amino acid solution (x100) (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.), and 0.1mM 2-mercaptoethanol (Thermo Fisher Scientific).
  • DMEM (high glucose) containing penicillin-streptomycin manufactured by Fuji Film Wako Pure Chemical Industries, Ltd.
  • the HEK293-derived Activin A-expressing extracellular vesicles obtained were added and cultured for 3 days in a static state in a CO 2 incubator (37° C., 5% CO 2 ).
  • recombinant Activin A protein manufactured by Shenandoah was added at the same concentration as the ELISA quantitative results of the HEK293-derived Activin A-expressing extracellular vesicles used, and cultured for 3 days.
  • the TaqMan probes shown in Table 10 both manufactured by Thermo Fisher Scientific were used for gene expression analysis, and the expression level of each gene was corrected by the expression level of GAPDH, a housekeeping gene. The results are shown in Table 11.
  • iPSC differentiation induction experiment using Activin A-expressing extracellular vesicles derived from HEK293 cells
  • Extracellular vesicles obtained by introducing the Mature Activin A vector into HEK293 cells were obtained in the same manner as in Condition 3 above.
  • the amount of extracellular vesicles added and the expression of differentiation markers were evaluated.
  • Human iPSCs (RPChiPS771 strain, manufactured by Repro Cell) were detached as single cells in the same manner as above.
  • the obtained cells were suspended in StemFit (registered trademark) medium supplemented with 10 ⁇ M Y-27632 (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) and placed in a 48-well plate coated with iMatrix-511 at 0.5 ⁇ g/cm 2 .
  • the cells were seeded at 2 ⁇ 10 4 cells/well and cultured in a static state in a CO 2 incubator (37° C., 5% CO 2 ) for 24 hours. (Day-1). On the day after seeding, it was confirmed that the cells had adhered, and the medium was replaced with a differentiation medium to start differentiation (Day 0).
  • HEK293-derived Mature Activin A-expressing extracellular vesicles whose Activin A concentration was determined by ELISA were added to the differentiation medium at concentrations of 0.05, 0.1, 0.2, and 0.5 ng/mL, and incubated for 3 days. Cultured. For comparison, Activin A recombinant protein (manufactured by Shenandoah) was added at concentrations of 0, 1, 10, and 100 ng/mL, and the medium was replaced every day in the control group. The results of evaluating gene expression 3 days after differentiation in the same manner as above are shown in FIG. 4.
  • mesodermal marker T and endodermal markers SOX17 and FOXA2 increased at low concentrations compared to the recombinant protein.
  • the endodermal marker T was highly expressed at the concentration of extracellular vesicles added, far exceeding the expression level of recombinant protein, suggesting that it may be particularly useful for differentiation into mesoderm. .
  • it is necessary to change the medium and add recombinant proteins each time during the 3-day differentiation period but in the case of extracellular vesicles, it is necessary to add recombinant proteins only on the first day without changing the medium. It was possible to induce iPSC differentiation.
  • Example 3 Effect on CAR-T cells 3.1. Preparation of plasmids 3.1.0.
  • CAR-P2A-Venus A polynucleotide (SEQ ID NO: 106) encoding a chimeric antigen receptor (CAR) (SEQ ID NO: 105) that recognizes Her2, a polynucleotide (SEQ ID NO: 108) encoding one of the 2A peptides, P2A (SEQ ID NO: 107).
  • CAR-P2A-Venus A polynucleotide (SEQ ID NO: 106) encoding a chimeric antigen receptor (CAR) (SEQ ID NO: 105) that recognizes Her2, a polynucleotide (SEQ ID NO: 108) encoding one of the 2A peptides, P2A (SEQ ID NO: 107).
  • a polynucleotide (SEQ ID NO: 112) encoding the artificially synthesized gene sequence CAR-P2A-Venus (SEQ ID NO: 111), which is composed of a polynucleotide (SEQ ID NO: 110) encoding Venus (SEQ ID NO: 109), is put into a pMX vector.
  • a vector for producing CAR-T cells was created. Since the 2A peptide sequence causes ribosome skipping, if a sequence encoding CAR-P2A-Venus is actually translated, an independent CAR-containing protein and an independent Venus-containing protein will be translated. 3.1.1.
  • Her2-MFGE8 Polynucleotide (SEQ ID NO: 80) encoding human Her2 signal peptide (SEQ ID NO: 79), polynucleotide encoding human Her2 (extracellular domain) (SEQ ID NO: 77) (SEQ ID NO: 78), peptide linker (SEQ ID NO: 81) ) (SEQ ID NO: 82) and a polynucleotide (SEQ ID NO: 84) encoding MFG-E8 (SEQ ID NO: 83), the artificially synthesized gene sequence Her2-MFG-E8 (SEQ ID NO: 85) ) was inserted into the pCAG-puro vector to create a vector for expressing Her2 on the membrane of extracellular vesicles.
  • Her2-MFGE8-IL-2 and Her2-MFGE8-IL-7 Gene sequence Her2-MFGE8- in which IL-2 (SEQ ID NO: 89) or IL-7 (SEQ ID NO: 93) is further fused to the C-terminus of Her2-MFG-E8 constructed above via a linker (SEQ ID NO: 87).
  • a polynucleotide (SEQ ID NO: 92 or 96) encoding IL-2 (SEQ ID NO: 91) or Her2-MFGE8-IL-7 (SEQ ID NO: 95), respectively, is inserted into a pCAG-puro vector, and Her2 and IL-2, or A vector was created to express Her2 and IL-7 on the membrane of extracellular vesicles.
  • Her2-CD81 A polynucleotide (SEQ ID NO: 98) encoding human Her2 (including a signal peptide, an extracellular domain, a transmembrane domain, and some intracellular domains) (SEQ ID NO: 97), and a polynucleotide encoding mouse CD81 (SEQ ID NO: 99)
  • Her2-CD81-IL-2 A sequence consisting of linker 8 (SEQ ID NO: 87), the sequence of IL-2 excluding the signal peptide, and linker 8 (SEQ ID NO: 87) was introduced into the 2nd loop of Her2-CD81 constructed above to create Her2-CD81-IL. -2 (SEQ ID NO: 103) was inserted into the pCAG-puro vector to create a vector for expressing Her2 and IL-2 on the membrane of extracellular vesicles. .
  • Her2-CD81-IL-15sa (super agonist) A polynucleotide encoding the N-terminal peptide (SEQ ID NO: 113) of TfR (transferrin receptor 1) (SEQ ID NO: 114); A polynucleotide (SEQ ID NO: 116) encoding linker 9 (SEQ ID NO: 115); A polynucleotide (SEQ ID NO: 118) encoding the IL-15R ⁇ sushi domain (SEQ ID NO: 117); A polynucleotide (SEQ ID NO: 120) encoding linker 10 (SEQ ID NO: 119); and a polynucleotide (SEQ ID NO: 122) encoding IL-15 (SEQ ID NO: 121) are linked in this order to form Her2-CD81- A polynucleotide (SEQ ID NO: 124) encoding IL-15sa (SEQ ID NO: 123) was prepared and inserted into the pCAG-puro vector,
  • hHer2-hCD81 It was created by replacing CD81 in 3.1.3 above with a human gene (amino acid sequence: SEQ ID NO: 129; polynucleotide sequence: SEQ ID NO: 130). 3.1.7. hHer2-hCD81-hIL-2 It was created by replacing CD81 and IL-2 in 3.1.4 above with human genes (amino acid sequence: SEQ ID NO: 131; polynucleotide sequence: SEQ ID NO: 132). 3.1.8. hCD19-hCD81 It was created by replacing Her2, the target factor (ie, antigen) in 3.1.6 above, with human CD19 (amino acid sequence: SEQ ID NO: 133; polynucleotide sequence: SEQ ID NO: 134). 3.1.9.
  • hCD19-hCD81-hIL-2 It was created by replacing Her2, the target factor (ie, antigen) in 3.1.7 above, with human CD19 (amino acid sequence: SEQ ID NO: 135; polynucleotide sequence: SEQ ID NO: 136).
  • hCD80-hCD9 In order to express human CD80, which is one of costimulatory molecules, on the membrane of extracellular vesicles, a polynucleotide (SEQ ID NO: 139) encoding a fusion protein (SEQ ID NO: 137) of human CD80 and human CD9, a tetraspanin, was used. ) was inserted into pCAG-puro or pMX vector to prepare a vector expressing the fusion protein.
  • FIG. 5 shows a schematic diagram of each gene structure, and Tables 12 to 14 show the sequence information.
  • the medium was changed 12 hours after transfection, and the supernatant was collected 60 hours after transfection and centrifuged at 300 g for 5 minutes. The collected supernatant was used as virus particles.
  • the recovered virus particles were spread on a plate coated with RetroNection according to the manufacturer's instructions. Lymph nodes removed from C57BL/6 mice were crushed on a 100 ⁇ m filter to obtain a lymph node cell suspension. 2 x 10 5 lymph node cells were suspended in 200 ⁇ L of RPMI 1640 medium supplemented with 10% fetal bovine serum, 50 ⁇ M 2-mercaptoethanol and penicillin/streptomycin, 10 ng/ml mIL-2 and cultured in Dynabeads Mouse according to the manufacturer's instructions.
  • T-Activator CD3/CD28 was added and cultured for 2 days. After culturing, the Dynabeads were removed, 2 ⁇ 10 5 cells were seeded on the plate coated with the virus particles, centrifuged at 500 g for 10 minutes, and cultured O/N to obtain CAR-T cells.
  • Lenti-X293T cells (TAKARA BIO) were seeded in a cell culture dish and cultured in Dulbecco's modified Eagle's medium supplemented with 2% fetal bovine serum and penicillin/streptomycin. .
  • the collected supernatant was used as virus particles.
  • the recovered virus particles were spread on a plate coated with RetroNection (TAKARA BIO) according to the manufacturer's instructions.
  • PBMC were separated from human peripheral blood using ficol, and 10 ng/m using Dynabeads TM Human T-Activator CD3/CD28 for T Cell Expansion and Activation (Thermo Fisher).
  • l's IL-2 Biolegend
  • the Dynabeads were removed, and 5 ⁇ 10 5 cells were seeded on the plate coated with the virus particles, centrifuged at 800 g for 30 minutes, and cultured for 2 days to obtain CAR-T cells.
  • HEK293T cells Preparation of extracellular vesicles containing Her2 molecules and T cell-stimulating cytokines in their membranes HEK293T cells were seeded in cell culture dishes and cultured in Dulbecco's modified Eagle's medium supplemented with 2% fetal bovine serum and penicillin/streptomycin. Cells at approximately 50% confluence were incubated with plasmids (Her2-CD81, Her2-CD81-IL-2, Her2-MFG-E8, Her2-MFG-E8-IL-2 or Her2-CD81-IL) according to the manufacturer's instructions.
  • plasmids Her2-CD81, Her2-CD81-IL-2, Her2-MFG-E8, Her2-MFG-E8-IL-2 or Her2-CD81-IL
  • pCAG vector encoding -15sa was transfected using Polyethylenenimine “Max” (manufactured by Polysciences).
  • the medium was changed 6 hours after transfection and 24 hours after transfection to Dulbecco's modified Eagle's medium supplemented with 2% fetal bovine serum and penicillin/streptomycin depleted of exosomes.
  • 72 hours after transfection the supernatant was collected, passed through a 0.22 ⁇ m filter, and then centrifuged at 300 g for 5 minutes. The supernatant was collected and centrifuged at 2,000g for 20 minutes. The supernatant was collected and centrifuged at 10,000g for 30 minutes. After collecting the supernatant and centrifuging it at 100,000 g for 2 hours, the supernatant was removed and the pellet was washed with PBS.
  • control exosomes or 293 exosomes were collected under similar conditions from HEK293 cells not transfected with the plasmid (hereinafter referred to as control exosomes or 293 exosomes).
  • 3.2.2.2 Preparation of extracellular vesicles containing human Her2 or CD19 molecules and T cell-stimulating cytokines in their membrane HEK293T cells were seeded in cell culture dishes and cultured in Dulbecco's modified Eagle's medium supplemented with 2% fetal bovine serum and penicillin/streptomycin. Cultured. Add plasmids (pCAG vector encoding hHer2-hCD81, hHer2-hCD81-hIL-2, hCD80-hCD9 or hCD19-hCD81, hCD19-hCD81-hIL-2) to cells at approximately 50% confluence according to the manufacturer's instructions.
  • control exosomes or 293 exosomes were collected under similar conditions from HEK293 cells not transfected with the plasmid (hereinafter referred to as control exosomes or 293 exosomes).
  • Extracellular small cells expressing Her2-CD81-IL-2 (approximately 3.8 times), Her2-CD81-IL-2 (approximately 8.2 times), or Her2-CD81-IL-15sa (approximately 7.4 times) cells significantly proliferated Venus-expressing CAR-T cells compared to controls.
  • Example 3.2.1.2. 7 ⁇ 10 4 Her2CAR-T cells prepared in Example 3.2 were suspended in 200 ⁇ L of X-VIVO 15 medium (Lonza) supplemented with 5% human serum and 10 mM neutralized N-acetyl L-Cysteine. .2.2.
  • extracellular vesicles hHer2-hCD81; hHer2-hCD81&hCD80-hCD9;hHer2-hCD81-hIL-2;hHer2-hCD81-hIL-2&hCD80-hCD9; or control extracellular vesicles (293 exosome) at a final concentration of 35 ⁇ g/
  • proliferation of Her2CAR-T cells was detected using a flow cytometer FACS Canto II (manufactured by BD Biosciences). The following antibodies were used for immunostaining (staining time: 15 minutes, temperature: 4°C).
  • Her2CAR-T cell proliferation was detected by Myc expression using a flow cytometer FACS Canto II (manufactured by BD Biosciences). ⁇ Alexa-fluor647 conjugate anti-human Myc antibody (manufactured by 9B11 Cell Signaling Technology) The results are shown in FIG. 7(B). Extracellular vesicles expressing Her2, CD80, and IL-2 significantly expanded Her2CAR-T cells.
  • extracellular vesicles (CD19-CD81; CD19-CD81 &CD80-CD9;CD19-CD81-IL-2; CD19-CD81-IL-2 & CD80-CD9) or control extracellular vesicles (293 exosome) at a final concentration of 35 ⁇ g/
  • proliferation of CAR-T cells was detected using a flow cytometer FACS Canto II (manufactured by BD Biosciences).
  • the following antibodies are used for immunostaining (staining time: 15 minutes, temperature: 4°C).
  • CAR-T cell proliferation was detected by GFP expression using a flow cytometer FACS Canto II (manufactured by BD Biosciences).
  • the results are shown in FIG. 7(C).
  • Extracellular vesicles expressing CD19, CD80, and IL-2 significantly expanded CD19CAR-T cells.
  • Table 15 below shows details of the sequence information used in the examples.

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Abstract

L'invention concerne [0] des vésicules extracellulaires qui présentent au moins une cytokine en extramembranaire; [1] des vésicules extracellulaires qui présentent au moins un facteur cible en extramembranaire; ou [2] des vésicules extracellulaires qui présentent au moins un facteur cible et au moins une cytokine en extramembranaire.
PCT/JP2023/016351 2022-04-25 2023-04-25 COMPOSITION POUR RÉGULER L'ACTIVATION DES CELLULES CAR-T, LA PROLIFÉRATION DES CELLULES SOUCHES HÉMATOPOÏÉTIQUES ET LA DIFFÉRENCIATION DES CELLULES iPS, ET SON UTILISATION WO2023210661A1 (fr)

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Citations (6)

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